Article

1-Anilino-8-naphthalene Sulfonate as a Protein Conformational Tightening Agent

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Abstract

1-Anilino-8-naphthalene sulfonate (ANS) anion is conventionally considered to bind to preexisting hydrophobic (nonpolar) surfaces of proteins, primarily through its nonpolar anilino-naphthalene group. Such binding is followed by an increase in ANS fluorescence intensity, similar to that occurring when ANS is dissolved in organic solvents. It is generally assumed that neither the negative sulfonate charge on the ANS, nor charges on the protein, participate significantly in ANS-protein interaction. However, titration calorimetry has demonstrated that most ANS binding to a number of proteins occurs through electrostatic forces, in which ion pairs are formed between ANS sulfonate groups and cationic groups on the proteins (D. Matulis and R. E. Lovrien, Biophys. J., 1998, Vol. 74, pp. 1-8). Here we show by viscometry and diffusion coefficient measurements that bovine serum albumin and gamma-globulin, starting from their acid-expanded, most hydrated conformations, undergo extensive molecular compaction upon ANS binding. As the cationic protein binds negatively charged ANS anion it also takes up positively charged protons from water to compensate the effect of the negative charge, and leaves the free hydroxide anions in solution thus shifting pH upward (the Scatchard-Black effect). These results indicate that ANS is not always a definitive reporter of protein molecular conformation that existed before ANS binding. Instead, ANS reports on a conformationally tightened state produced by the interplay of ionic and hydrophobic characters of both protein and ligand.

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... Binding of 8-Anilino-1-napthalenesulfonic acid (ANSA) to mLOX was studied by excitation at 370 nm and emission spectra were recorded from 400 to 600 nm 30 . mLOX (3 μM) was treated with ANSA in 1:100 ratio and incubated at 37°C for 30 min and the spectrum was obtained. ...
... ANSA is a chemical molecule which binds to the exposed hydrophobic patches in a protein surface 30 . Recombinant mLOX showed an emission maximum at 450 nm. ...
Article
Lysyl oxidase (LOX), a promising therapeutic target for the progression of cancer and fibrosis, has not been well characterized yet. A major difficulty faced in LOX characterization is its lack of solubility in common buffers. In this study, mature LOX (mLOX) was cloned, purified and its purity was ascertained by mass spectroscopy. Through screening various buffers, 0.2 M glycine-NaOH buffer with 10% glycerol pH 8.0 was identified to maintain mLOX in its soluble state. About 67% of the refolded mLOX was found to be in copper bound state after His-tag removal. Catalytic properties Km and kcat were found to be 3.72 × 10−4 M and 7.29 ×103s−1. In addition, collagen cross-linking in ARPE-19 cells was augmented on exposure to mLOX, endorsing its biological activity. Circular Dichroism revealed that mLOX comprises 8.43% of α-helix and 22% of β-strand and it was thermally stable up to 90°C. Disulfide linkage imparts the structural stability in LOX which was experimentally ascertained with intrinsic and extrinsic fluorescence studies.
... Binding of 8-Anilino-1-napthalenesulfonic acid (ANSA) to mLOX was studied by excitation at 370 nm and emission spectra were recorded from 400 to 600 nm 30 . mLOX (3 μM) was treated with ANSA in 1:100 ratio and incubated at 37°C for 30 min and the spectrum was obtained. ...
... ANSA is a chemical molecule which binds to the exposed hydrophobic patches in a protein surface 30 . Recombinant mLOX showed an emission maximum at 450 nm. ...
Article
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Lysyl oxidase (LOX), a promising therapeutic target for the progression of cancer and fibrosis, has not been well characterized yet. A major difficulty faced in LOX characterization is its lack of solubility in common buffers. In this study, mature LOX (mLOX) was cloned, purified and its purity was ascertained by mass spectroscopy. Through screening various buffers, 0.2 M glycine-NaOH buffer with 10% glycerol pH 8.0 was identified to maintain mLOX in its soluble state. About 67% of the refolded mLOX was found to be in copper bound state after His-tag removal. Catalytic properties K m and k cat were found to be 3.72 × 10 −4 M and 7.29 ×10 3 s −1. In addition, collagen cross-linking in ARPE-19 cells was augmented on exposure to mLOX, endorsing its biological activity. Circular Dichroism revealed that mLOX comprises 8.43% of α-helix and 22% of β-strand and it was thermally stable up to 90°C. Disulfide linkage imparts the structural stability in LOX which was experimentally ascertained with intrinsic and extrinsic fluorescence studies.
... Confirmation of PfAdoMetDC fold using ANS fluorescence-based assay: The ANS binds to hydrophobic (nonpolar) surfaces of proteins, through its nonpolar anilinonaphthalene group (50). For this reason, ANS is used to estimate the levels of exposed hydrophobic clusters of protein. ...
... In addition, the effect of nucleotides on the conformation of each Hsp70 was investigated by repeating the assay in the absence or presence of 5 mM ATP/ADP. Similarly, biophysical characterization of PfAdoMetDC was conducted by monitoring both intrinsic (tryptophan and /tyrosine); and extrinsic (1-anilinonapthelene-8sulfonate, ANS; Sigma Aldrich, Darmstadt, Germany) emission spectra as previously described (50). Since PfAdoMetDC possesses a single tryptophan residue and 33 tyrosine residues, the intrinsic PfAdoMetDC emission spectra was monitored at 300 -400 nm. ...
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Hsp70 is one of the most prominent molecular chaperones. Although Hsp70s from various organisms are generally conserved, they exhibit specialised cellular functions. It remains to be fully understood how these highly conserved molecules exhibit specialised functional features. Plasmodium falciparum Hsp70-1 (PfHsp70-1) is a cytosol localised molecular chaperone that is implicated in the cyto-protection and pathogenicity of the malaria parasite. In the current study, we investigated the comparative structure-function features of PfHsp70-1 relative to its homologue, E. coli Hsp70 (DnaK) and a chimeric protein, KPf, that was constituted by the ATPase domain of DnaK and the substrate binding domain (SBD) of PfHsp70-1. Recombinant forms of all the three Hsp70s exhibited similar secondary and tertiary structural fold. We further established that compared to DnaK, both KPf and PfHsp70-1 were more stable to heat stress and exhibited higher basal ATPase activity. A recombinant P. falciparum Hsp40 (PfHsp40) stimulated the ATPase activities of all the three Hsp70s. In addition, both PfHsp70-1 and KPf exhibited preference for asparagine rich peptides as opposed to DnaK. Furthermore, all the three proteins exhibited self-association capabilities in vitro. Recombinant P. falciparum adenosylmethionine decarboxylase (PfAdoMetDC) co-expressed in E. coli with either KPf or PfHsp70-1 was produced as a fully folded product. On the other hand, co-expression of PfAdoMetDC with heterologous DnaK in E. coli did not promote folding of the former. These findings demonstrated that the SBD of PfHsp70-1 regulates several functional features of the protein and that this molecular chaperone is tailored to facilitate folding of plasmodial proteins.
... The ANS is a well-known extrinsic fluorescent dye that emits significantly more fluorescence due to the hydrophobicity of a binding position and the constrained mobility of ANS, as a result of structural changes and when attaching to protein clumps [5,32]. ANS binding with all the samples mentioned above was evaluated by using the previously published method [33]. ANS concentration used was 5 μM. ...
... Furthermore, CC, which is a major protein oxidation biomarker, was assessed during the process of glycation. CC is generated during the EGPs or Amadori product for-mation and contributes to the AGE accretion in tissues [4,33]. The scavenging of carbonyl compounds by natural and synthetic agents has been shown to be potent method for inhibition of nonenzymatic glycation reaction [4,5]. ...
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The current study is aimed at studying the inhibitory effect of glycyrrhizic acid (GA) on D-ribose-mediated protein glycation via various physicochemical analyses and in silico approaches. Being a potent free radical scavenger and a triterpenoid saponin, GA plays a vital role in diminishing the oxidative stress and thus could be an effective inhibitor of the nonenzymatic glycation process. Our data showed that varying concentrations of GA inhibited the in vitro BSA-AGEs via inhibiting the formation of fructosamines, fluorescent AGEs, scavenging protein carbonyl and hydroxymethyl furfural (HMF) content, and protection against D-ribose-induced modification of BSA as evident by increased free Arg and Lys residues in GA-treated Gly-BSA samples. Moreover, GA also attenuated D-ribose-induced alterations in the secondary structure of BSA by protecting the α-helix and β-sheet conformers and amide-I band delocalization. In addition, GA attenuated the modification in β-cross amyloid structures of BSA and in silico molecular interaction study too showed strong binding of GA with higher number of Lys and Arg residues of BSA and binding energy (ΔG) of -8.8 Kcal/mol, when compared either to reference standard aminoguanidine (AG)-BSA complex (ΔG: -4.3 Kcal/mol) or D-ribose-BSA complex (ΔG: -5.2 Kcal/mol). Therefore, GA could be a new and favorable inhibitor of the nonenzymatic glycation process that ameliorates AGEs-related complications via attenuating the AGE formation and glycation-induced multiple protein modifications with a reduced risk of adverse effects on protein structure and functionality; hence, it could be investigated at further preclinical settings for the treatment and management of diabetes and age-associated complications.
... ANS has a low quantum fluorescence yield in polar media, which increases significantly upon transition to a non-polar medium [27]. Interaction of ANS with other molecules occurs through non-polar anilinonaphthalene and negatively charged sulphonate groups [28] (Fig. 3a). ...
... Structures of 8-anilino-1-naphthalenesulfonic acid (a) and Fura 2 (b)[28,29] Fig. 4 Dependence of λ max a the absorbance spectra and I max b the fluorescence emission Fura 2 on Cu-NFs and Fe 3+ -Cu-NFs. Initial [Fura 2] = 60 µmol/L (recalculation in ...
Article
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Among nanomaterials, we can now distinguish a special class called nanoflowers (NFs). These new nanostructures have aroused the interest of scientists due to the topographic features of nanolayers, the special location which allows a higher surface-to-volume ratio compared to classical spherical nanoparticles, thereby significantly increasing the efficiency of surface reactions for nanoflowers. The main value of nanoflowers is their action as enzyme stabilizers. A protein stability is usually enhanced by immobilization on a nanoflower surface through charge affinity and covalent bonds. The possibility of their use in vivo in biocatalysis, biosensors and medicine has been also investigated. We now report on the synthesis of two different nanoflowers: Cu nanoflowers and Fe3+ attached Cu nanoflowers and their interaction with two fluorescent probes, anilino-1-naphthalenesulfonic acid (ANS) and Fura 2, and two proteins, human serum albumin (HSA) and thrombin. Nanoflowers did not bind ANS, but bind efficiently to Fura 2 and both proteins. Modification of Cu–NFs by Fe3+ leads to significant changes in their binding capacity to fluorescent probe Fura 2 and both proteins. Their ability to bind fluorescent probe Fura 2 increased eightfold, and their ability to bind HSA and thrombin increased five times. Regarding Fe3+–Cu–NFs, a difference in binding between HSA and thrombin was found that can be explained by their structural features. Our data indicate the possibility of using studied nanoflowers for sorption of fluorescent probes and proteins.
... In addition, the effect of nucleotides on the conformation of each Hsp70 was investigated by repeating the assay in the absence or presence of 5 mM ATP/ADP. Similarly, biophysical characterization of PfAdoMetDC was conducted by monitoring both intrinsic (tryptophan and /tyrosine) and extrinsic (1-anilinonapthelene-8-sulfonate, ANS; Sigma Aldrich) emission spectra as previously described [39]. Since PfAdoMetDC possesses a single tryptophan residue and 33 tyrosine residues, the intrinsic PfAdoMetDC emission spectra were monitored at 300-400 nm. ...
... The ANS binds to hydrophobic (nonpolar) surfaces of proteins, through its nonpolar anilino-naphthalene group [39]. For this reason, ANS is used to estimate the levels of exposed hydrophobic clusters of protein. ...
Article
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Hsp70 is a conserved molecular chaperone. How Hsp70 exhibits specialized functions across species remains to be understood. Plasmodium falciparum Hsp70-1 (PfHsp70-1) and Escherichia coli DnaK are cytosol localized molecular chaperones that are important for the survival of these two organisms. In the current study, we investigated comparative structure-function features of PfHsp70-1 relative to DnaK and a chimeric protein, KPf, constituted by the ATPase domain of DnaK and the substrate binding domain (SBD) of PfHsp70-1. Recombinant forms of the three Hsp70s exhibited similar secondary and tertiary structural folds. However, compared to DnaK, both KPf and PfHsp70-1 were more stable to heat stress and exhibited higher basal ATPase activity. In addition, PfHsp70-1 preferentially bound to asparagine rich peptide substrates, as opposed to DnaK. Recombinant P. falciparum adenosylmethionine decarboxylase (PfAdoMetDC) co-expressed in E. coli with either KPf or PfHsp70-1 was produced as a fully folded product. Co-expression of PfAdoMetDC with heterologous DnaK in E. coli did not promote folding of the former. However, a combination of supplementary GroEL plus DnaK improved folding of PfAdoMetDC. These findings demonstrated that the SBD of PfHsp70-1 regulates several functional features of the protein and that this molecular chaperone is tailored to facilitate folding of plasmodial proteins.
... the environment of these residues changes. Alternatively, the fluorescence of a solvatochromic probe such as ANS (1,8-anilinonaphthalene sulfonate) (Anderson and Weber, 1969;Slavik, 1982;Matulis and Lovrien, 1998;Matulis et al., 1999;Cimmperman and Matulis, 2011) or Sypro Orange (Lo et al., 2004;Niesen et al., 2007) can be followed as the probe binds to the exposed hydrophobic residues upon protein unfolding. FTSA is a less-widely used technique than ITC, but its use has significantly increased when compound libraries are screened to determine the best binders to target proteins (Pantoliano et al., 2001;Yanchunas et al., 2005;McDonnell et al., 2009). ...
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The aim of rational drug design is to develop small molecules using a quantitative approach to optimize affinity. This should enhance the development of chemical compounds that would specifically, selectively, reversibly, and with high affinity interact with a target protein. It is not yet possible to develop such compounds using computational (i.e., in silico) approach and instead the lead molecules are discovered in high-throughput screening searches of large compound libraries. The main reason why in silico methods are not capable to deliver is our poor understanding of the compound structure–thermodynamics and structure–kinetics correlations. There is a need for databases of intrinsic binding parameters (e.g., the change upon binding in standard Gibbs energy (ΔGint), enthalpy (ΔHint), entropy (ΔSint), volume (ΔVintr), heat capacity (ΔCp,int), association rate (ka,int), and dissociation rate (kd,int)) between a series of closely related proteins and a chemically diverse, but pharmacophoric group-guided library of compounds together with the co-crystal structures that could help explain the structure–energetics correlations and rationally design novel compounds. Assembly of these data will facilitate attempts to provide correlations and train data for modeling of compound binding. Here, we report large datasets of the intrinsic thermodynamic and kinetic data including over 400 primary sulfonamide compound binding to a family of 12 catalytically active human carbonic anhydrases (CA). Thermodynamic parameters have been determined by the fluorescent thermal shift assay, isothermal titration calorimetry, and by the stopped-flow assay of the inhibition of enzymatic activity. Kinetic measurements were performed using surface plasmon resonance. Intrinsic thermodynamic and kinetic parameters of binding were determined by dissecting the binding-linked protonation reactions of the protein and sulfonamide. The compound structure–thermodynamics and kinetics correlations reported here helped to discover compounds that exhibited picomolar affinities, hour-long residence times, and million-fold selectivities over non-target CA isoforms. Drug-lead compounds are suggested for anticancer target CA IX and CA XII, antiglaucoma CA IV, antiobesity CA VA and CA VB, and other isoforms. Together with 85 X-ray crystallographic structures of 60 compounds bound to six CA isoforms, the database should be of help to continue developing the principles of rational target-based drug design.
... A, An example of thermal denaturation curves of CA XII (upper panel, strong binding) and CA I (lower panel, weak binding) in the presence of increasing concentrations of compound 27b. The protein unfolding curves were registered by following the fluorescence of 8-anilino-1-naphthalene sulfonate[50] in the buffer containing 50 mM sodium phosphate (pH 7.0), 100 mM sodium chloride and 2.0% (v/ v) DMSO. The midpoint temperature of the unfolding transition is equal to melting temperature (T m ): high-affinity ligand increases T m of the protein to a much greater extent as the concentrations of ligand increase than the low-affinity ligand. ...
Article
By applying an approach of a “ring with two tails”, a series of novel inhibitors possessing high-affinity and significant selectivity towards selected carbonic anhydrase (CA) isoforms has been designed. The “ring” consists of 2-chloro/bromo-benzenesulfonamide, where the sulfonamide group is as an anchor coordinating the Zn(II) in the active site of CAs, and halogen atom orients the ring affecting the affinity and selectivity. First “tail” is a substituent containing carbonyl, carboxyl, hydroxyl, ether groups or hydrophilic amide linkage. The second “tail” contains aryl- or alkyl-substituents attached through a sulfanyl or sulfonyl group. Both “tails” are connected to the benzene ring and play a crucial role in selectivity. Varying the substituents, we designed compounds selective for CA VII, CA IX, CA XII, or CA XIV. Since due to binding-linked protonation reactions the binding-ready fractions of the compound and protein are much lower than one, the “intrinsic” affinities were calculated that should be used to study correlations between crystal structures and the thermodynamics of binding for rational drug design. The “intrinsic” affinities together with the intrinsic enthalpies and entropies of binding together with co-crystal structures were used demonstrate structural factors determining major contributions for compound affinity and selectivity.
... At the same time, we experimentally prove that ANS binding to all investigated types of amyloids accompanying by the significantly increase in the probe fluorescence intensity. The assumption that ANS binding does not require pre-existing hydrophobic sites in protein molecules to start dye-protein binding reaction is in a good agreement with early works [50,52]. Results of these works indicate that the protein does not behave as a static particle when ANS binds to it. ...
Article
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Fluorescent probes thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) are widely used to study amyloid fibrils that accumulate in the body of patients with serious diseases, such as Alzheimer’s, Parkinson’s, prion diseases, etc. However, the possible effect of these probes on amyloid fibrils is not well understood. In this work, we investigated the photophysical characteristics, structure, and morphology of mature amyloid fibrils formed from two model proteins, insulin and lysozyme, in the presence of ThT and ANS. It turned out that ANS affects the secondary structure of amyloids (shown for fibrils formed from insulin and lysozyme) and their fibers clusterization (valid for lysozyme fibrils), while ThT has no such effects. These results confirm the differences in the mechanisms of these dyes interaction with amyloid fibrils. Observed effect of ANS was explained by the electrostatic interactions between the dye molecule and cationic groups of amyloid-forming proteins (unlike hydrophobic binding of ThT) that induce amyloids conformational changes. This interaction leads to weakening repulsion between positive charges of amyloid fibrils and can promote their clusterization. It was shown that when fibrillogenesis conditions and, consequently, fibrils structure is changing, as well as during defragmentation of amyloids by ultrasonication, the influence of ANS to amyloids does not change, which indicates the universality of the detected effects. Based on the obtained results, it was concluded that ANS should be used cautiously for the study of amyloid fibrils, since this fluorescence probe have a direct effect on the object of study.
... ANS is a fluorescent dye used to study the surface hydrophobicity and conformational changes in protein molecules. It specifically binds to the protein's hydrophobic clusters and shows an increase in fluorescence intensity upon binding to them (Matulis et al., 1999). ...
Article
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Purified soya bean proteins (glycinin and conglycinin) are known to form amyloid‐like aggregates in vitro at a higher temperature. Soya beans (chunks) are textured proteinaceous vegetables made from defatted soya flour by heating it above 100°C and extruding under high pressure. Therefore, it was assumed that subjecting the soya bean proteins to high temperatures raises the possibility of forming amyloids or amyloid‐like protein aggregates. Hence, the present study aimed to examine the presence of amyloid‐like protein aggregates in soya beans. The isolated protein aggregates from hydrated soya beans displayed typical characteristics of amyloids, such as the red shift in the absorption maximum (λmax) of Congo red (CR), high Thioflavin T (ThT), and 8‐Anilinonapthalene‐1‐sulfonate (ANS) binding, and fibrilar morphology. Furthermore, these aggregates were found to be stable against proteolytic hydrolysis, confirming the specific property of amyloids. The presence of amyloid‐like structures in soya beans raises concerns about their implications for human nutrition and health. Practical applications Protein aggregation has usually been considered detrimental. The traditional food‐processing conditions, such as thermal processing, are associated with protein denaturation and aggregation. The formation of ordered protein aggregates with extensive β‐sheet are progressively evident in various protein‐rich foods known as amyloid, which expands food safety concerns. Instead, it is also associated with poor nutritional characteristics. The present study concerns the presence of amyloid‐like protein aggregates in widely consumed native soya beans, which are manufactured by extensive heat treatment of defatted soy flour. Although there is no indication of their toxicity, these aggregates are found to be proteolytically resistant. The seminal findings in this manuscript suggest that it is time to adapt innovative food processing and supplementation of bioactive molecules that can prevent the formation of such protein aggregates and help maximize the utilization of protein‐based nutritional values.
... Extrinsic fluorescence dyes like ANS are well known to emit significantly greater fluorescent when exposed to more hydrophobic environments including protein's interior apolar surfaces due to structural changes as well as when combines with protein aggregates [40]. ANS binding with unmodified and Gly-BSA in presence or absence of ID and AG (1, 5, 25 and 50 μM) was assessed following the standard protocol [41]. The samples were excited at 380 nm and the emission spectra were recorded in the wavelength range of 400-600 nm on Agilent Cary Eclipse Spectro-fluorimeter. ...
Article
Non-enzymatic protein glycation results in the formation of advanced glycation end products (AGEs) leads to the pathogenesis of long-term diabetic complications. Iridin (ID), an antioxidant, plays an important role in protecting against oxidative stress and could therefore be an efficacious anti-glycating regimen. Herein, we assessed the anti-glycating potential of ID against d-ribose induced glycation of bovine serum albumin (BSA) by various biophysical and biochemical techniques. Our results from several physicochemical assays advocated that ID was able to evidently prevent the AGEs generation via reducing hyperchromicity, early glycation products (EGPs), carbonyl content (CC), hydroxymethyl furfural (HMF) content, production of fluorescent AGEs, protection against loss of secondary structure (i.e. α-helix and β-sheets) of proteins, increasing the free lysine and free arginine content, reduced binding of congo red (CR), and reduced thioflavin T (ThT) and 8-aninilo-1-napthalene sulphonate (ANS)-specific fuorescence in glycated-BSA (Gly-BSA). On the basis of these findings, we concluded that ID possesses the significant anti-glycation potential and may be established as a remarkable anti-AGEs therapeutic agent. Further in-vivo and clinical studies are still warranted to uncover the therapeutic effects of ID against age-related as well as metabolic diseases.
... Therefore, the microenvironment of Trp and Tyr residues of RG did not change during the process of binding between RG and β-CN. ANS anion was conventionally considered to bind to preexisting hydrophobic surfaces of proteins (Matulis, Baumann, Bloomfield, & Lovrien, 1999). After the surface hydrophobic region of the RG was occupied by the fluorescent probe ANS, the binding between β-CN and RG still occurred (Fig. 4E&F), while the maximum absorption wavelength of RG was redshifted (Fig. 4F). ...
Article
The present study investigated the interaction between acid casein and rice glutelin (RG) under neutral conditions. Large particle populations contain casein-RG complex which consisted of β-casein, αs1-casein, and RG was formed. The results of multispectral studies indicated that the peptide chain and secondary structure of casein did not change during the binding process. Besides, the binding behaviours seemed to be closely related to the surface hydrophobic region of RG. Thermodynamic parameter calculations showed that the main driving forces behind this structure formation were the hydrogen bonds and van der Waals forces. On the other hand, the addition of RG caused the generation of small particle populations (∼20 nm) which mainly composed of κ-casein and αs2-casein, and such small particle populations maintained aggregation through residual calcium. Moreover, it is noting that these two particle populations can coexist stably, this finding will provide an avenue to produce, by design, casein nanoparticles.
... Also the position of the emission maximum is sensitive to polarity, and a blue shift is observed when polarity of the environment decreases (the position of peak emission maximum is 50 nm lower in ethanol compared to H 2 O). ANS has been used to probe the hydrophobic domains in proteins and membranes [52][53][54], hydrogels [55], and to follow the phase transi- tions of thermoresponsive polymers as PDMEAMA [56], PNIPAm-b-PIL [57] and PEO-b-PNIPAm [58]. ...
Article
Soft poly(N-vinylcaprolactam) (PNVCL) based nanogels were prepared and surface-decorated with gold nanoparticles (AuNPs). The applicability of the hybrid nanogels (PNVCL-AuNPs) as carriers for low molar mass substances was of special interest. AuNPs protected with a mixture of 11-azidoundecanothiol and 11-mercaptoundecanoic acid were bound to propargyl functionalized PNVCL based nanogels. Acidic groups on the surfaces of AuNPs and PNVCL based nanogels stabilize the particle dispersions against precipitation above the phase transition temperature of PNVCL. Both the neat PNVCL nanogels and the PVCL-AuNPs shrink upon heating the dispersions. Even though the AuNPs are mainly located in the soft surface layer of the nanogels, the PNVCL-AuNPs respond to visible light as well as to radio-frequency (RF) irradiation by shrinking due to the AuNPs acting as nanoheaters. Interactions of linear PNVCL, PNVCL nanogels and PNVCL-AuNPs with two fluorescent probes were studied as function of increasing temperature. Once bound to the polymer the fluorescent probe may or may not be released from it, depending on its polarity and water solubility. Presence of AuNPs changed the release behavior of the water soluble charged fluorescent probe from the nanogels.
... Quantification of protein aggregates can be carried out by a plethora of techniques, like HPLC, western blotting, microscopy, capillary electrophoresis, analytical ultracentrifugion, or UV-Vis spectroscopy, but characterisation of the aggregates normally requires a specific technique, such as circular dichroism, fluorescence, infrared, Raman spectroscopy, or nuclear magnetic resonance (Mahler et al., 2009). Recently, folding probes have started to be implemented as an alternative method (Matulis et al., 1999;Liu et al., 2014;Sagle et al., 2004). ...
Article
Protein aggregation is normally associated with amyloidosis, namely motor neurone, Alzheimer’s, Parkinson’s or prion diseases. However, recent results have unveiled a concept of gradual increase of protein aggregation associated with the ageing process, apparently not necessarily associated with pathological conditions. Given that protein aggregation is sufficient to activate stress-response and inflammation, impairing protein synthesis and quality control mechanisms, the former is assumed to negatively affect cellular metabolism and behaviour. In this review the state of the art in protein aggregation research is discussed, namely the relationship between pathology and proteostasis. The role of pathology and ageing in overriding protein quality-control mechanisms, and consequently, the effect of these faulty cellular processes on pathological and healthy ageing, are also addressed.
... Similarly, Fig. 5C shows the fluorescence spectra of ANS which reflects the surface hydrophobicity of protein. The fluorescence intensity of whole rice glutelin containing more JRG was significantly reduced, which meant that modification of JRG can reduce the hydrophobicity of whole rice glutelin (Matulis, Baumann, Bloomfield, & Lovrien, 1999). To sum up, the results of the interfacial properties measurement showed that the addition of JRG can bury hydrophobic amino acid residues with relatively high abundance on the surface, such as Trp, Tyr, and Phe residues (Fig. S1) during the pH-cycle process. ...
... During the FTSA experiments, the protein solution was heated from room temperature to 99 °C while following the extrinsic fluorescence of a solvatochromic probe, such as 1,8-anilinonaphthalene sulfonate (ANS), that strongly changes its fluorescence upon protein unfolding (Stryer 1965;Slavik et al. 1982;Matulis and Lovrien 1998;Matulis et al. 1999). The thermal melting temperatures (T m ) are strongly dependent on the ligand binding affinity and concentration as previously described (Brandts and Lin 1990;Matulis et al. 2005;Cimmperman et al. 2008). ...
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Membrane-associated carbonic anhydrase (CA) isoform IV participates in carbon metabolism and pH homeostasis and is implicated in the development of eye diseases such as retinitis pigmentosa and glaucoma. A series of substituted benzenesulfonamides were designed and their binding affinity to CA IV was determined by fluorescent thermal shift assay and isothermal titration calorimetry (ITC). Compound [(4-chloro-2-phenylsulfanyl-5-sulfamoyl-benzoyl)amino]propyl acetate (19) bound CA IV with the Kd of 1.0 nM and exhibited significant selectivity over the remaining 11 human CA isoforms. The compound could be developed as a drug targeting CA IV. Various forms of recombinant CA IV were produced in Escherichia coli and mammalian cell cultures. Comparison of their temperature stability in various buffers and salt solutions demonstrated that CA IV is most stable at slightly alkaline conditions and at elevated sodium sulfate concentrations. High-resolution X-ray crystallographic structures of ortho-Cl and meta-thiazole-substituted benzene sulfonamide in complex with CA IV revealed the position of and interactions between the ligand and the protein. Sulfonamide inhibitor binding to CA IV is linked to several reactions—the deprotonation of the sulfonamide amino group, the protonation of CA–Zn(II)-bound hydroxide at the active site of CA IV, and the compensating reactions of the buffer. The dissection of binding-linked reactions yielded the intrinsic thermodynamic parameters, characterizing the interaction between CA IV and the sulfonamides in the binding-able protonation forms, including Gibbs energy, enthalpy, and entropy, that could be used for the characterization of binding to any CA in the process of drug design.
... Blue dots represent extrinsic fluorescence probes (e.g., ANS molecules): with rays-fluorescent probes, without rays-nonfluorescent probes. When temperature is increased, the hydrophobic surfaces of a protein become accessible to ANS and this results in increased fluorescence intensity of the probe binding [9] and that such a probe may significantly affect the protein structure [10]. However, the mechanism of ANS binding to proteins is still not fully understood, but the probe is one of the best reporters of the protein unfolding. ...
Chapter
In this chapter we describe the model that quantifies interactions between proteins and ligands applying the thermal shift assay. When combined with fluorescence-based measurements of protein thermal unfolding, this model forms the basis of the fluorescent thermal shift assay—a widely applicable and cost-effective technique to quantify ligand affinity towards proteins. Most ligands stabilize proteins against thermal denaturation and shift their melting points towards higher temperatures. Equations that relate the shift in protein melting temperature with the ligand concentration are presented. The assay has been used to determine affinities of various sulfonamide inhibitor binding to carbonic anhydrase isoforms. The results illustrate applicability and limitations of the fluorescent thermal shift assay.
... These include cylinders, 25,29,31,32 spherocylinders, 32 spindle shapes, 32 double cones, 32 Platonic solids, 30,33 red-blood-cell shapes, 34 hollow spherical caps, 25 microwedges, 35 dumbbell shapes, 25,27 chains of spheres, 27,33,36,37 three-body swimmers consisting of Platonic solids, 30 a microswimmer that propels itself by a rotating helical flagellum, 30 oligomers, 27,33 and macromolecules. 26,28,33,[38][39][40][41] Besides direct numerical calculations, experimental data from observing and analyzing a particle's orientation-resolved trajectories can be used to deter-mine its hydrodynamic resistance matrix. 9,42 However, the shapes for which this matrix is known constitute only a very small amount of the particle shapes that can be synthesized and that are relevant for research. ...
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The hydrodynamic resistance matrix is an important quantity for describing the dynamics of colloidal particles. This matrix encodes the shape- and size-dependent hydrodynamic properties of a particle suspended in a simple liquid at low Reynolds number and determines the particle's diffusion tensor. For this reason, the hydrodynamic resistance matrix is typically needed when modeling the motion of free purely Brownian, externally driven, or self-propelled colloidal particles or the behavior of dilute suspensions of such particles on the basis of Langevin equations, Smoluchowski equations, classical dynamical density functional theory, or other appropriate methods. So far, however, the hydrodynamic resistance matrix was available only for a few particle shapes. In this article, we therefore present the hydrodynamic resistance matrices for various particle shapes that are relevant for current research, including apolar and polar as well as convex and partially concave shapes. The elements of the hydrodynamic resistance matrices are given as functions of shape parameters like the aspect ratio of the corresponding particle so that the results apply not only to discrete but instead to continuous sets of particle shapes. This work shall stimulate and support future studies on colloidal particles with anisometric shapes.
... In addition to the identification and characterization of hydrophobic binding sites of proteins, ANS has also been found to induce partial folding to the acid denatured states of Cytochrome C and Pectate lyase C [20,21]. Furthermore, ANS also acts to induce stability and functions as a conformation tightening agent as its binding to the acid expanded and most hydrated conformations of bovine serum albumin and gamma-globulin leads to the extensive molecular compaction [22,23]. ...
Article
The exposed hydrophobic patches of protein are widely detected through the binding by the fluorescent probes such as 1-anilino-8-naphthalene sulfonate (ANS), Nile Red (NR) and 1-(N-phenylamino) naphthalene, N-(1-Naphthyl) aniline (1NPN). Interestingly, at pH 4, where the Toxoplasma gondii Ferredoxin-NADP(+) reductase (TgFNR) is stable, an exclusive binding and fluorescence emission was observed for ANS. To understand the underlying difference in the binding of ANS, NR and 1NPN; their effect on the protein structure was studied in detail. ANS was found to interact with TgFNR via electrostatic as well as hydrophobic interactions at pH 4. NR and 1NPN did not show any such binding to TgFNR in the similar conditions, however showed strong hydrophobic interaction in the presence of NaCl or DSS (2, 2-dimethyl-2-silapentane-5-sulfonate). The subsequent structural studies suggest that ANS, NaCl and DSS induced partial unfolding of TgFNR by modulating ionic interactions of the enzyme, leading to the exposure of buried hydrophobic patches amicable for the binding by NR and 1NPN. The induced unfolding of TgFNR by ANS is unique and thus cautions to use the fluorescent dye as simple indicator to probe the exposed hydrophobic patches of the protein or its folding intermediates.
... An analysis of the surface hydrophobicity of β-LG was carried out using the fluorescent probe ANS to monitor plasma-induced structural alterations. Conventionally, ANS is considered to bind to pre-existing hydrophobic sites on the protein surface through its non-polar anilinonaphthalene group and this increases the ANS fluorescence intensity (Matulis, Baumann, Bloomfield, & Lovrien, 1999). A substantial increase in fluorescence intensity was observed after 1 min of plasma treatment (Fig. 4a), indicating an increase in the hydrophobicity of β-LG due to additional hydrophobic sites being exposed on the protein surface. ...
Article
The present study investigated the effects of dielectric-barrier-discharge (DBD) plasma treatment at 1, 2, 3, and 4 min on the reduction of the IgG binding capacity of β-lactoglobulin (β-LG). The IgG binding capacity of β-LG was reduced by 58.21% following a plasma treatment time of 4 min as confirmed by western-blot and ELISA analyses. The reduction in IgG binding capacity of β-LG was directly related to a stepwise change in its structure. The initial drop in the IgG binding capacity of β-LG was found to be caused by conformational alteration, free sulfhydryl exposure and cross-linkage of molecules induced by oxidation of NH-/NH2-functional groups of peptide bonds and of sensitive amino acid residues (Tyr, Trp) as confirmed by SDS-PAGE, surface hydrophobicity and multi-spectroscopic analyses. Plasma treatment therefore has great potential as a substitute treatment for enzymatic hydrolysis for the production of hypoallergenic milk protein-based products.
... c Determined T m values from curves in part b are then plotted as a function of added ligand concentration (filled circles). The line is drawn according to the model, enabling the affinity (K d ) calculation Matulis et al. 1999;Cimmperman and Matulis 2011) and SYPRO Orange (Lo et al. 2004;Niesen et al. 2007). In our experience, ANS exhibits more favorable properties for FTSA than SYPRO Orange because ANS binds weaker to proteins than SYPRO Orange. ...
Article
A standard operating procedure for a fluorescence-based thermal shift assay (FTSA) is provided describing its typical applications, advantages and limitations. FTSA is a simple, robust, universal and quick assay to determine protein–ligand binding affinities and protein stabilities in the presence of various excipients and solution conditions. Therefore, the assay is very useful for the straightforward characterization of new recombinantly produced proteins. The assay has a wide dynamic range enabling simultaneous determination of affinities in the milimolar to picomolar range. The assay could be used for essentially any protein that is sufficiently soluble and stable in the studied aqueous solution. Here we provide examples and typical experimental protocols for both affinity and stability determinations.
... ANS is basically non-fluorescent in aqueous media but shows strong fluorescence upon binding to the hydrophobic pocket of HSA. [15][16][17] Thus, the replacement of ANS bound to HSA with ligands causes quenching of the ANS fluorescence. It has been reported that subdomain IIIA and IIA of HSA have the binding pockets of ANS to provide fluorescent property to ANS. 18,19 Therefore, the fluorescence quenching is caused mainly by the competition with the ANS bound to the IIIA/IIA subdomain. ...
Article
Therapeutic peptides and diagnostic agents with their molecular size below the renal clearance threshold suffer from short blood circulation time. Here, we report a novel design of peptide-based ligand with a strong binding affinity to human serum albumin (HSA), which can be used as a tag to extend the blood circulation of small-size molecules. We designed ligands with dual alkyl groups connected with a negatively charged spacer. The ligands showed both higher binding affinity to HSA and a higher retention in mice blood than that of a single alkylated peptide.
... Reduction in F max might be due to reduced binding of ANS to SNaCas. SNaCas had high electronegative charge on the surface and ANS is an anionic fluorescent dye (Matulis, Baumann, Bloomfield, & Lovrien, 1999), which may have resulted in lower interaction of ANS to SNaCas, hence reduction in F max was observed. C. Gupta et al. ...
... Upon binding of the probe to accessible hydrophobic regions of proteins, an increase in fluorescence is observed, which is used as a measure of protein surface hydrophobicity. Oxidative stress induced changes in hydrophobic patches of isolated HSA was studied by ANS binding [52]. The ANS dye mixed with HSA isolated from T1DM subjects showed an increase in fluorescence intensity suggesting oxidative stress induced exposure of hydrophobic patches (Fig. 8). ...
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Chronic oxidative stress fuels pathogenesis of a large set of diseases. Oxidative stress is the cause and consequence of numerous diseases including type 1 diabetes mellitus (T1DM), in which there is selective destruction of insulin producing pancreatic β-cells. Studies have documented that hyperglycemia produces profound stress. In vivo production of numerous reactive oxygen, nitrogen, chlorine species and lipid/sugar oxidation products are result of persistent hyperglycemia. Post-translational modifications by reactive species may create new antigenic epitopes and play a role in the development of autoimmune response. In this paper our main focus was to establish the effect of existing hyperglycemia induced oxido-nitrosative stress in T1DM patients on the integrity of human serum albumin. Raised nitric oxide, carbonyl, RBC hemolysis, lowered ferric reducing antioxidant power (FRAP), thiol and deformed RBC in T1DM are all highly suggestive of persistent oxido-nitrosative stress. Hyperglycemia induced generation of advanced glycation end products (AGEs) was established by LCMS. Chronic oxido-nitrosative stress can modify HSA in T1DM patients, producing immunologically active albumin. Therefore, it is speculated that the aberrant HSA may play a role in the initiation/progression of T1DM.
... 3.3.2 | Effects of pH and NaCl concentration on the tertiary structure of buffalo β-CN by ANS Binding of ANS with nonpolar regions of protein leads to a blueshift to its maximum fluorescence and rise of its fluorescence intensity (Matulis et al., 1999). The strong affinity of ANS to proteins in the "molten globule" state is caused by the lack of tertiary structure F I G U R E 5 Tryptophan in trinsic fluorescence spectra of buffalo β-CN (a), λmax (b), surface hydrophobicity index (c) as a function of NaCl concentration (0.01, 0.05, 0.08, 0.1, 0.2, and 0.3 M) at pH 7.0 F I G U R E 6 The relative fluorescence intensity of buffalo β-CN (a), λmax (b), surface hydrophobicity index (c) treated by pH (pH 7.5, 7.0, 6.5, 6.0, 5.5, and 5.0) in 10 mM sodium phosphate buffer using 1-anilinonaphthalene-8sulfonic acid probe (ANS) F I G U R E 7 The relative fluorescence intensity of buffalo β-CN (a), λmax (b), surface hydrophobicity index (c) treated by various NaCl concentration (0.01, 0.05, 0.08, 0.1, 0.2, and 0.3 M) at pH 7.0, in 10 mM sodium phosphate buffer using 1-anilinonaphthalene-8sulfonic acid probe (ANS) (Semisotnov et al., 1991). ...
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In the present study, we aimed to investigate the effects of pH and sodium chloride (NaCl) concentration on the structure of β‐casein (β‐CN) purified from buffalo milk using circular dichroism (CD), intrinsic tryptophan, and anilino‐8‐naphthalene sulfonate (ANS) fluorescence spectroscopy. We found that NaCl concentration played a critical role in the stability of the secondary structure of β‐CN. The CD negative peak had a redshift as the NaCl concentration was increased and accompanied by a decrease of β‐sheet content and an increase of α‐helix content. ANS fluorescence spectroscopy also indicated that higher NaCl concentration and lower pH significantly affected the tertiary structure of β‐CN. Dynamic light scattering (DLS) results showed that the particle size of buffalo β‐CN had a blueshift, and then a redshift within the pH range of 5.0–7.5, and it showed a redshift when the NaCl concentration was increased. This study aimed to investigate the effects of pH and NaCl concentration on purified β‐casein structure from buffalo milk using circular dichroism (CD), intrinsic tryptophan and ANS fluorescence spectroscopic. It was found that NaCl concentration was a critical factor in affecting stability of the secondary structure of β‐casein.
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Silver nanoparticles (SNPs) have been increasingly used in medicines and biomaterials as a drug carriers and diagnostic or therapeutic material due to their smaller size, large surface area and cell penetration ability. Here we report the preparation of SNPs of diameter 10 ± 3 nm by using silver nitrate and sodium borohydride and the interaction of synthesized SNPs with our model protein β-lactoglobulin (β-lg) in 10 mM phosphate buffer at pH 7.5 after thermal exposure at 75 °C. Heat exposed β-lg forms amyloidal fibrillar aggregates whereas this protein aggregates adopt rod-like shape instead of fibrillar structure in presence of SNP under the same conditions. Size of the synthesized SNPs is confirmed by UV–Visible spectroscopy, SEM and TEM. Interactions and subsequent formation of molecular assembly of heat stressed β-lg with SNP were investigated using Th-T assay and ANS binding assay, DLS, RLS, CD, FT-IR, SEM, TEM. Docking study parallely also support the experimental findings.
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A desire to replicate the structural and functional complexity of proteins with structured, sequence‐specific oligomers motivates study of the structural features of water‐soluble peptoids (N‐substituted glycine oligomers). Understanding the molecular‐level details of peptoid self‐assembly in water is essential to advance peptoids' application as novel materials. Peptoid 1, an amphiphilic, putatively helical peptoid previously studied in our laboratory, shows evidence of self‐association in aqueous solution. In this work, we evaluate how changes to aqueous solution conditions influence the self‐association of 1. We report that changes to pH influence the fluorescence and CD spectroscopic features as well as the peptoid's interaction with a solvatochromic fluorophore and its apparent size as estimated by size exclusion chromatography. Addition of guanidine hydrochloride and ammonium sulfate also modulate spectroscopic features of the peptoid, its interaction with a solvatochromic fluorophore, and its elution in size exclusion chromatography. These data suggest that the ordering of the self‐assembly changes in response to pH and with solvent additives and is more ordered at higher pH and in the presence of guanidine hydrochloride. The deeper understanding of the self‐association of 1 afforded by these studies informs the design of new stimuli‐responsive peptoids with stable tertiary or quaternary structures.
Patent
Przedmiotem wynalazku jest nowy przewodzący polimer bisbitiofenowy, wdrukowany moleku-larnie za pomocą białek, w tym ludzkiej albuminy, sposób jego przygotowania i jego zastosowanie jako warstwy rozpoznającej czujnika elektrochemicznego do selektywnego wykrywania i/lub oznacza-nia ludzkiej albuminy w moczu i we krwi.
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The extrapolation of hepatic clearance (CL) from data determined in an in vitro assay in the absence of albumin (ALB) to that in the presence of ALB in liver in vivo were often inaccurate using traditional in vitro-to-in vivo extrapolation (IVIVE) methods for drugs binding to the ALB. It is recognized that considering an ALB-facilitated hepatic uptake phenomenon in the IVIVE can improve the extrapolation. Therefore, the present study provides a comparison of two existing models that account for the ALB-facilitated hepatic uptake phenomenon in the IVIVE of CL. These models assume an interaction of the ALB-bound drug complex with the hepatocyte membrane that enhanced the dissociation of the drug from ALB to result in increased unbound intracellular drug levels available for metabolism or transporter-mediated elimination. One model refers to the old facilitated-dissociation model (FDM), which is based on a binding isotherm and necessitates knowing the specific input parameters of the interaction (i.e., relative capacity of the interaction, dissociation constant, number of binding sites and ALB concentration). The other model is based on the same theory, but is recent and more speculative although it presumes that each interaction between the ALB-drug complex and the hepatocyte surface would at all times enhance and deliver the corresponding dissociated bound drug into the hepatocytes, and, hence, has the advantage to use less binding information. Consequently, this model simply consists of adjusting the unbound fraction determined in plasma in vitro of each drug (fup-adjusted) with the real differential of ALB concentration between the plasma and liver in vivo to estimate the corresponding differential of ALB-drug complex also assumed available to deliver the unbound drug moiety for hepatic uptake in vivo versus in vitro. Application of these two models significantly improved the IVIVEs of CL of drugs, and, hence, the next step was to compare these two models with the same dataset. Recently published data on the hepatic uptake of two organic anions, namely, 1-anilino-8-naphthalene sulfonate (ANS) and pitavastatin (PTV) provide all binding informations. As expected, the results indicate that these two models (FDM and fup-adjusted) are conceptually and mathematically equivalent as well as they successfully predicted the experimentally-determined ratios of the unbound intrinsic CL (CLint) in the presence of ALB in vivo to that in the absence of ALB in vitro. However, these models were equivalent particularly for PTV because its ALB-drug complex showed a relevant capacity of interaction and dissociation with the hepatocyte membrane. Conversely, for ANS, the model of fup-adjustedoverestimated the ratio of unbound CLintby contrast to the FDM model because its ALB-drug complex demonstrated a significantly lower capacity of interaction with the membrane. The rational is simply because the model of fup-adjustedpresumably assumed an important facilitated-uptake phenomenon for each drug, whereas the FDM model was derived from binding data specific to each drug. Overall, these two models are complementary and all contribute towards achieving the same objective of quantifying the ALB-facilitated uptake phenomenon; however, the FDM model is more specific but its application necessitates collecting more binding data compared to the model of fup-adjustedthat can be used prospectively to predict the maximal effect resulting from the facilitated-uptake in IVIVE.
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The hydrophobic Aβ peptide is highly aggregation prone; it first forms soluble oligomers, which then convert into the amyloid fibrils found in the cerebral plaques of Alzheimer's disease. It is generally understood that as the peptide concentration of Aβ increases, the fibrillization process is accelerated, but we examine the limits on this phenomenon. We found that once a threshold concentration of Aβ is exceeded, a stable oligomer is formed at the expense of fibril formation. The suppression of fibril formation was observed by amyloid-binding dye Thioflavin T and solution nuclear magnetic resonance (NMR). Small-angle X-ray scattering, size exclusion chromatography, and analytical ultracentrifugation demonstrated that Aβ peptides form a range of compact species, with a dimer being an early highly populated oligomer. Solution NMR allowed us to define the secondary structure of this Aβ dimer, which shows interlocking contacts between C-terminal peptide strands. Thus, we present a novel Aβ oligomer that resists conversion to fibrils and remains stable for more than one year.
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The influence of irradiation on protein- porphyrin complexes at various spectral ranges was studied. Limited applicability of evaluating protein damage degree in terms of fluorescence quenching of the tryptophan protein residues was shown. Depending on the conditions of photoirradiation and the way of porphyrins interaction with bovine serum albumin (BSA), photoirradiation of the porphyrin-protein complexes may result in proteolysis or photolinking inside the protein globule, and in some cases, in destruction of the complex itself. P1 5-(4′-N-tertbutyloxycarbonylglicinaminophenyl)-10,15,20-triphenylporphine, unlike 5,10,15,20-tetraphenylporphin (TPP), has fixed localization in the protein due to “anchor” group providing the stability of the photoinduced transformations of a protein globule. For all porphyrin-protein systems studied, irradiation by light at the Soret band range for P1-protein systems leads to proteolysis of BSA globule, while the irradiation by light with the wavelength more than 500 nm, does not result in total protein damage, but causes oxidation of susceptible amino acid residues. The measurement of the hydrodynamic radii of a protein globule before and after irradiation provided objective information on the proceeding photoinduced processes (proteolysis and crosslinking) which can be used for predicting the mechanism of the cell damage (necrosis, autophagy, apoptosis).
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Background: Glycosylation is an effective method to modify protein. However, there is a lack of research on the property changes of glycosylated protein during storage. In the present study, the changes in the physicochemical, functional, and structural properties of xylo-oligosaccharide (XOS) glycosylated egg white powder (EWP) (XOS-EWP conjugates) prepared with different glycosylation conditions (XOS/EWP ratio and reaction time) were investigated when stored at 25 °C and 60% relative humidity. Results: In the 12 weeks of storage, the degree of grafting, browning, and the formation of Maillard reaction products of XOS-EWP conjugates increased. The increase in XOS/EWP ratio and reaction time led to an increase in protein aggregation, though a decrease in solubility, due to increased degree of glycosylation and structural changes. Furthermore, improved gel hardness of XOS-EWP conjugates deteriorated, while improved emulsification ability was kept stable during storage. For the sample with a lower XOS/EWP ratio and reaction time, the gel hardness and emulsifying properties underwent little or no deterioration even improving during storage. The results could be attributed to the limited degree of glycosylation, further unfolding of the protein structure, increased surface hydrophobicity of protein, and improved thermal characteristics. Conclusion: During storage, the Maillard reaction would continue to occur in the glycosylated EWP, further affecting the performance of modified EWP. Modified EWP prepared under different glycosylation conditions performed differently during storage. Modified EWP with a larger XOS/EWP ratio and reaction time meant it was harder to maintain good performance. Modified EWP with a smaller XOS/EWP ratio and reaction time changed significantly to better performances. © 2022 Society of Chemical Industry.
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Reported here are the design and fluorescence characters of a NiII/TbIII polymer, [Tb2Ni3(HCAM)6(H2O)12] n (1) (H3CAM = chelidamic acid). Under physiological conditions, the binding of biocompatible water soluble 1 to human serum albumin (HSA) was studied by spectroscopy techniques, which revealed that 1 could inherent the fluorescent light of HSA in a static quencher course and change the HSA second-level structure. The marked enhancement in 1 and its fluorescence intensity provide conclusive evidence that 1 can play the role of a "turn-on" sensor for recognition and detection of HSA in other biological interferents with a Ksv value of 7.68 × 104 M-1 and a detection limit of 0.14 μM. Luminescence experiments show that 1 has high selectivity and sensitivity to MnO4- in other anions. Its quenching efficiency (Ksv) is 5.54 × 103 M-1, and the detection limit is 0.29 μM.
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Differential scanning calorimetry (DSC) determines the enthalpy change upon protein unfolding and the melting temperature of the protein. Performing DSC of a protein in the presence of increasing concentrations of specifically-binding ligand yields a series of curves that can be fit to obtain the protein–ligand dissociation constant as done in the fluorescence-based thermal shift assay (FTSA, ThermoFluor, DSF). The enthalpy of unfolding, as directly determined by DSC, helps improving the precision of the fit. If the ligand binding is linked to protonation reactions, the intrinsic binding constant can be determined by performing the affinity determination at a series of pH values. Here, the intrinsic, pH-independent, affinity of acetazolamide binding to carbonic anhydrase (CA) II was determined. A series of high-affinity ligands binding to CAIX, an anticancer drug target, and CAII showed recognition and selectivity for the anticancer isozyme. Performing the DSC experiment in buffers of highly different enthalpies of protonation enabled to observe the ligand unbinding-linked protonation reactions and estimate the intrinsic enthalpy of binding. The heat capacity of combined unfolding and unbinding was determined by varying the ligand concentrations. Taken together, these parameters provided a detailed thermodynamic picture of the linked ligand binding and protein unfolding process.
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Otolin-1 is a C1q family member and a major component of the organic matrix of fish otoliths and human otoconia. To date, the protein molecular properties have not been characterized. In this work, we describe biochemical characterization and comparative studies on saccular-specific otolin-1 derived from Danio rerio and Homo sapiens. Due to the low abundance of proteins in the otoconial matrix, we developed a production and purification method for both recombinant homologues of otolin-1. Danio rerio and Homo sapiens otolin-1 forms higher-order oligomers that can be partially disrupted under reducing conditions. The presence of Ca²⁺ stabilizes the oligomers and significantly increases the thermal stability of the proteins. Despite the high sequence coverage, the oligomerization of Danio rerio otolin-1 is more affected by the reducing conditions and presence of Ca²⁺ than the human homologue. The results show differences in molecular behaviour, which may be reflected in Danio rerio and Homo sapiens otolin-1 role in otolith and otoconia formation.
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Among the twelve catalytically active carbonic anhydrase isozymes present in the human body, the CAIX is highly overexpressed in various solid tumors. The enzyme acidifies the tumor microenvironment enabling invasion and metastatic processes. Therefore, many attempts have been made to design chemical compounds that would exhibit high affinity and selective binding to CAIX over the remaining eleven catalytically active CA isozymes to limit undesired side effects. It has been postulated that such drugs may have anticancer properties and could be used in tumor treatment. Here we have designed a series of compounds, methyl 5-sulfamoyl-benzoates, which bear a primary sulfonamide group, a well-known marker of CA inhibitors, and determined their affinities for all twelve CA isozymes. Variations of substituents on the benzenesulfonamide ring led to compound 4b, which exhibited an extremely high observed binding affinity to CAIX; the Kd was 0.12 nM. The intrinsic dissociation constant, where the binding-linked protonation reactions have been subtracted, reached 0.08 pM. The compound also exhibited more than 100-fold selectivity over the remaining CA isozymes. The X-ray crystallographic structure of compound 3b bound to CAIX showed the structural position, while several structures of compounds bound to other CA isozymes showed structural reasons for compound selectivity towards CAIX. Since this series of compounds possess physicochemical properties suitable for drugs, they may be developed for anticancer therapeutic purposes.
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Disulfide bond formed between the cysteine pairs plays a key role in maintaining the integrity of the protein structure and function. The ubiquitin-associated (UBA) domain of human HOIP contains three cysteine residues, Cys⁵⁰⁴, Cys⁵⁵¹, and Cys⁵⁷². Disulfide bonds formed by Cys⁵⁰⁴ and Cys⁵⁵¹ residues are highly conserved, but the effect of disulfide bonds on the biochemical characteristics of UBA has not been elucidated. In addition, due to the presence of isolated Cys⁵⁷², inactive inclusion bodies may be formed during protein expression or trigger protein aggregation during protein purification. In this study, the co-expression of SUMO fusion protein combined with SUMO protease (ULP enzyme) in Escherichia coli was successfully applied to improve the soluble expression of UBA domain. Introduced three mutants (UBAC551A, UBAC572A and UBAC551,572A) determined the effects of disulfide bonds on the biochemical characteristics of UBA. Circular dichroism and analytical size exclusion chromatography results showed that the target proteins obtained by co-expression could be folded correctly and had biological activity. Both thermal-induced and urea-induced results demonstrated that the elimination of disulfide bonds would significantly reduce the stability of UBA. Fluorescence spectroscopy result showed that the elimination of disulfide bonds slightly increases the binding affinity of UBA to ligands. In summary, soluble, stable and active UBA domain and its mutants were prepared by co-expression system, which will further contribute to the structural and functional research of UBA.
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The in vitro-to-in vivo extrapolation (IVIVE) methods for predicting the hepatic clearance (CL) of drugs based on microsomal or hepatocyte data have certainly advanced; however, there is still place for improving the extrapolations using in vitro assays containing no plasma proteins. Accordingly, there is a discussion on whether the free drug hypothesis or an albumin (ALB)-mediated hepatic uptake phenomenon is the best scaling method. Therefore, the objectives of this study were to guide the prediction of CL and to diagnose which scaling method between the free drug hypothesis and ALB-mediated uptake could be more accurate; this, irrespective of the mechanism(s) governing CL if the drugs can get to the hepatocyte membrane. The analysis of several datasets demonstrated that almost all values of CL in vivo fall within the two calculated values of CL use as boundaries from: 1) the free drug hypothesis, and 2) ALB-mediated uptake. The average value from these two CL boundaries predicted the CL in vivo with incredible accuracy. Validating these boundaries in preclinical species prior going to human as well as considering the fractional binding in plasma increased the accuracy. Overall, this study is another step towards guiding the CL prediction in drug discovery and development.
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Qualitative and quantitative understanding of partially folded states of protein is essential in gaining deeper insights into folding pathways. We have observed a partially folded state of bovine serum albumin (BSA) in the presence of 2,2,2-trifluoroethanol at pH11.2 which does not resembles the properties of the molten globule state. ThT, a frequently used marker for protein fibrils have two order of greater affinity towards the intermediate state at pH11.2 compared to native BSA at pH7.4. Surprisingly, the binding of ANS with this partially folded state is weaker than that of native state of BSA. Combined fluorescence, circular dichroism spectroscopy and isothermal titration calorimetric studies indicate that for such partially folded state, ThT is a better marker compared to ANS. The results have highlighted the importance of dyes like ThT in characterizing partilally folded states of protein which might appear as intermediates in the funnel moldel describing the protein folding pathway.
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The present study reports the multi-technique results of the interaction of a series of bile salts, sodium cholate (NaC), sodium taurocholate (NaTC), sodium deoxycholate (NaDC), and sodium taurodeoxycholate (NaTDC) with Trypsin under the experimental conditions of 25 °C and pH 7.0. The interactions between Trypsin and the bile salts were characterized by the surface tension measurements and various spectroscopic techniques like UV–Visible absorption, steady-state fluorescence, and circular dichroism. The results of surface tension measurements reveal a strong interaction of Trypsin (50 μM) with the increasing concentration of bile salts, being higher with the bile salt of greater hydrophobicity. The critical aggregation concentration of bile salts in the presence of Trypsin (C1) showed that the bile salts interact strongly with the Trypsin in the order of NaTDC > NaDC > NaTC > NaC. UV–visible, steady-state fluorescence, and circular dichroism spectroscopic results confirmed significant unfolding of Trypsin due to its interaction with the bile salts, the extent of which followed the same sequence as observed in the surface tension results. It could be concluded that the hydrophobic bile salts that show lower C1 values and have less delocalized charge, are more effective in unfolding the Trypsin. The study would help to understand the hydrophobicity-driven unfolding of proteins aided by the biological surfactants like bile salts and can help to devise efficient proteolytic enzyme-based detergent formulations and understand the role of such amphiphiles as antimicrobial agents.
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Background Carbonic anhydrases (CAs) regulate pH homeostasis via the reversible hydration of CO2, thereby emerging as essential enzymes for many vital functions. Among 12 catalytically active CA isoforms in humans, CA IX has become a relevant therapeutic target because of its role in cancer progression. Only two CA IX inhibitors have entered clinical trials mostly due to low affinity and selectivity properties. Objective The current review presents the design, development, and identification of the selective nano- to picomolar CA IX inhibitors VD11-4-2, VR16-09, and VD12-09. Methods and Results Compounds were selected from our database, composed of over 400 benzensulfonamides, synthesized at our laboratory, and tested for their binding to 12 human CAs. Here we discuss the CA CO2 hydratase activity/inhibition assay and several biophysical techniques, such as fluorescent thermal shift assay and isothermal titration calorimetry, highlighting their contribution to the analysis of compound affinity and structure-activity relationships. To obtain sufficient amounts of recombinant CAs for inhibitor screening, several gene cloning and protein purification strategies are presented, including site-directed CA mutants, heterologous CAs from Xenopus oocytes, and native endogenous CAs. The cancer cell-based methods, such as clonogenicity, extracellular acidification, and mass spectrometric gas-analysis are reviewed, confirming nanomolar activities of lead inhibitors in intact cancer cells. Conclusions Novel CA IX inhibitors are promising derivatives for in vivo explorations. Furthermore, the simultaneous targeting of several proteins involved in proton flux upon tumor acidosis and the disruption of transport metabolons might improve cancer management.
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Amyloids, a well-ordered β-sheet-enriched structural network, can be broadly defined as insoluble protein aggregates that are linked to a wide variety of diseases including systemic amyloidosis and some neurodegenerative disorders. Ferulic acid (FA), a phenolic acid, abundant in antioxidant and efficient pharmaceutical has beneficial effects against several ailments. Based on this, we have investigated the protective role of FA on amyloid formation of bovine β-lactoglobulin (β-lg), a model globular protein. Using a set of in vitro biophysical methods, such as UV-Vis spectroscopy, fluorescence, circular dichroism, transmission electron microscopy, etc., our research group has concluded that FA significantly inhibits the heat-induced amyloid formation of β-lg and this inhibitory effect is dose-dependent. Exposed surface hydrophobicity of β-lg amyloid fibrils decreased significantly in the presence of FA. Docking study revealed that ionic and hydrogen bonding interactions between FA and β-lg prevented protein conformational changes leading to fibrillation. We anticipate that our finding would give an insight into the protein aggregation inhibited by the antioxidant compound, FA and pave the way for finding and developing other new small molecules (protein misfolding inhibitors) that give similar result against amyloid fibril formation and its allied neurodegenerative disorders. Ferulic acid inhibits the aggregation of β-lg in a dose-dependent manner.
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Divalent metal cations can play a role in protein aggregation diseases, including cataract. Here we compare the aggregation of human γS-crystallin, a key structural protein of the eye lens, via mutagenesis, UV light damage, and the addition of metal ions. All three aggregation pathways result in globular, amorphous structures that do not elongate into fibers. We also investigate the molecular mechanism underlying copper (II)-induced aggregation. This work was motivated by the observation that zinc (II)-induced aggregation of γS-crystallin is driven by intermolecular bridging of solvent-accessible cysteine residues, while in contrast, copper (II)-induced aggregation of this protein is exacerbated by the removal of solvent-accessible cysteines via mutation. Here we find that copper (II)-induced aggregation results from a complex mechanism involving multiple interactions with the protein. The initial protein-metal interactions result in the reduction of Cu(II) to Cu(I) with concomitant oxidation of γS-crystallin. In addition to the intermolecular disulfides that represent a starting point for aggregation, intramolecular disulfides also occur the cysteine loop, a region of the N-terminal domain that was previously found to mediate the early stages of cataract formation. This previously unobserved ability of γS-crystallin to transfer disulfides intramolecularly suggests that it may serve as an oxidation sink for the lens after glutathione levels have become depleted during aging. γS-crystallin thus serves as the last line of defense against oxidation in the eye lens, a result that underscores the chemical functionality of this protein, which is generally considered to play a purely structural role.
Thesis
Many therapeutic proteins, currently in development or manufacture, are expressed as inclusion bodies. Proteins must be refolded from the inclusion body into its active form. This refolding step can be difficult to develop and can give a low yield. This project was designed, in partnership with Avecia Biologies, with the purpose of improving the way in which commercial protein refold steps are developed in order to reduce development costs, manufacturing costs and time-to-market of new protein pharmaceuticals. Development of protein refold steps for commercial manufacture was investigated. Methods for quantifying refolding yield were evaluated. It was determined that refold development experiments could best be improved by decreasing the amount of material required for experiments, increasing throughput of the experiments and designing the experiment efficiently. Techniques for performing refold reactions in microwells were developed and their usefulness was demonstrated with experiments to improve a refold step for IGF-1. Experiments using only 60mg of protein gave a refold step yield of 50% (up from 26%) and a 4-fold decrease in refold volume, using 20%v/v propylene glycol, 0.25M arginine, 0.25M GdnHCI as refold buffer additives. Techniques were developed for performing rapid refold development experiments using laboratory automation, in particular a pipetting robot. The efficiency of automated microwell refold reactions was demonstrated with refold optimisation experiments, using lysozyme as an example protein. These experiments, which used only 130mg of protein and could be performed by one scientist in just two days, identified refold conditions giving a 58% increase in step yield. Another automated microwell-scale refold experiment using trypsinogen as the example protein established (in 1 day, using 3.1 mg protein) that a 13% increase in yield could be achieved by including 20mM PEG300 and lOmM pentanol in the refold buffer. The application of these techniques in a commercial bioprocess development laboratory was further considered.
Article
The design and construction of artificial light-harvesting systems for solar energy conversion to chemical energy has been an active research field. A variety of molecules and materials have been used to mimic the function of the light-harvesting antenna. However, the improvement or regulation of the antenna effect of the existing artificial light-harvesting systems is less explored. Coordination polymers have aroused extensive concern due to their applications in light-harvesting and energy conversion. Herein, it is found that silver ion can dramatically enhance the emission of dye encapsulated in the coordination polymer nanoparticles (CPNs). The mechanism of Ag⁺-induced fluorescence enhancement is elucidated. Taking advantage of the effect of Ag⁺ ions, the regulation of CPN-based light-harvesting system by Ag⁺ is achieved for the first time. The antenna effect could be up to 2.3 times the original value by adding Ag⁺ ions. The present work provides a new approach to regulate the antenna effect of the light-harvesting system with the advantages of convenience, rapidity, low cost, and flexibility.
Article
Nucleobindin-2 (Nucb2) is a widely expressed multi-domain protein. Nucb2 participates in many physiological processes, i.e. calcium level maintenance, feeding regulation in the hypothalamus, emotion and stress regulation, and many others. To date, this protein has not been structurally characterized. We describe the first comparative structural analysis of two homologs, a Gallus gallus and a Homo sapiens Nucb2. The in silico analysis suggested that apo-Nucb2s contain a mosaic-like structure, consisting of intertwined disordered and ordered regions. Surprisingly, the hydrogen-deuterium exchange mass spectrometry results revealed that Nucb2 is divided into two parts: an N-terminal half with a stable mosaic-like structure and a disordered C-terminal half. However, the presence of Ca²⁺ induces the formation of a mosaic-like structure in the C-terminal half of the Nucb2s. The Ca²⁺ also affects the tertiary and quaternary structure of Nucb2s. The presence of Ca²⁺ leads to an overall compaction of the Nucb2 molecule, resulting in structural change that is propagated along the molecule, which in turn affects the quaternary structure of the protein. Intrinsic disorder, and the mosaic-like Ca²⁺ dependent structure of Nucb2s, might be seen as the molecular factors responsible for their multifunctionality. Thus, Nucb2s might function as the versatile Ca²⁺ sensor involved in signal transduction.
Article
The ANS− (1-anilino-8-naphthalene sulfonate) anion is strongly, dominantly bound to cationic groups of water-soluble proteins and polyamino acids through ion pair formation. This mode of ANS− binding, broad and pH dependent, is expressed by the quite rigorous stoichiometry of ANS− bound with respect to the available summed number of H+ titrated lysine, histidine, and arginine groups. By titration calorimetry, the integral or overall enthalpies of ANS− binding to four proteins, bovine serum albumin, lysozyme, papain, and protease omega, were arithmetic sums of individual ANS−–polyamino acid sidechain binding enthalpies (polyhistidine, polyarginine, polylysine), weighted by numbers of such cationic groups of each protein (additivity of binding enthalpies). ANS− binding energetics to both classes of macromolecules, cationic proteins and synthetic cationic polyamino acids, is reinforced by the organic moiety (anilinonaphthalene) of ANS−. In a much narrower range of binding, where ANS− is sometimes assumed to act as a hydrophobic probe, ANS− may become fluorescent. However, the broad overall range is sharply dependent on electrostatic, ion pair formation, where the organic sulfonate group is the major determinant of binding.
Article
Bovine serum albumin exists in a compact form between pK 4.3 and 10.5. Below pH 4.3 and above pH. 10.5 it undergoes expansion. This paper shows that the compact form is held together by a network of bonds involving the side-chain groups of the protein. Near pH 4.2 or 10.5 the titration of some of these side chains results in transition to an expandable form. This form undergoes continuous expansion, increasing with charge and decreasing with ionic strength, so as to reduce the electrostatic free energy. A further slow molecular change occurs on standing. The entire process, including the final slow change, is reversible.
Article
Hydrogen ion titration curves of de-ionized bovine serum albumin have been obtained at four ionic strengths and two temperatures. They have been analyzed by equations treating the electrostatic effect as an empirical factor. The ionizing groups found present and their intrinsic pK's at 25° are: 1 α-carboxyl (3.75 assumed), 99 side chain carboxyl (3.95), 16 imidazole (6.9), 1 α-amino (7.75 assumed), 57 ε-amino (9.8), 19 phenolic (10.35) and 22 guanidyl (>12). The pK values found for carboxyl, amino and phenolic groups are abnormal : they suggest that these groups all participate in some kind of internal bonding, such that the carboxylate ion is favored over the un-ionized COOH group, the neutral NH2 group over the NH3+ ion, and the un-ionized phenolic groups over the phenolate ion. The heats and entropies of ionization are incompatible with carboxylate-phenolic hydrogen bonds previously suggested. Empirical values of the electrostatic interaction factor w are independent of pH between pH 4.3 and 10.5, but fall off sharply outside this region. Thus serum albumin appears to maintain constant shape and size between pH 4.3 and 10.5, but it must expand outside this region. This conclusion is in agreement with data obtained by other methods.
Article
Measurements have been made of the viscosity of solutions of bovine serum albumin, as a function of concentration, pH (i.e., molecular charge) and ionic strength. Between pH 4.3 and 10.5 the results may be interpreted on the basis of a compact, sparingly hydrated, undeformable protein molecule, not greatly different from a sphere. At 25°, over a considerable range of pH and ionic strength near the isoionic point, the intrinsic viscosity is 0.037, which is about 50% greater than would be expected for a sphere with 20% hydration and about equal to what would be expected for a prolate ellipsoid with the same hydration and with an axial ratio of about 3:1. (However, evidence is presented to indicate that the albumin molecule is, in fact, not a rigid ellipsoid.) As the molecular charge is increased by approaching the limits of the pH range here covered, a small increase in intrinsic viscosity occurs, which is of the order of magnitude predicted for rigid spheres by Booth's equation for the electroviscous effect. This equation also accounts for a small increase at the isoionic point in the complete absence of salt. The effect of concentration is shown to be a sum of two effects, one independent of charge or ionic strength, the other an electrostatic effect increasing roughly as the square of the charge and decreasing with ionic strength.
Article
The diffusion coefficient of bovine serum albumin (BSA) was measured in aqueous solutions of varying temperature, pH, BSA concentration, and ionic strength. The measurements were carried out using dynamic light scattering with the photon detector set at a 90-degrees angle. The measured diffusion coefficients were compared to calculated values using phenomenological models which account for the screened Coulomb interaction between the charged proteins, as well as hydrodynamic corrections to the friction factor. The dimensions of BSA were obtained from structural data, and the charge on the protein was estimated using titration data. Although the measured and calculated values of the diffusion coefficient are in general agreement, significant discrepancies are observed. No single theoretical model seems capable of accurate predictions for all ranges of ionic strength and protein concentration.
Article
This chapter discusses the quasi-elastic light scattering (QLS) that can be used in enzymology, and in other areas of molecular biology and macromolecular chemistry, to study the dynamics of macromolecules. Its predominant use has been to measure translational diffusion coefficients, which can be interpreted in terms of Stokes radii and combined with sedimentation coefficients in the Svedberg equation to yield molecular weights. Other applications are experimentally more difficult, and therefore less common, though they may be promising in favorable cases. These include the measurement of rotational diffusion coefficients of nonspherical scatterers, conformational changes (e.g., helix-coil transitions), the kinetics of biomolecular reactions, the measurements of intramolecular flexibility, molecular motion in gels, bacterial motility, muscular contraction, electrophoretic mobility, protoplasmic streaming, the conduction of nerve impulses, and the flow of blood and other biological fluids.
Article
Data from differential scanning calorimetry (DSC) may be used to estimate very large binding constants that cannot be conveniently measured by more conventional equilibrium techniques. Thermodynamic models have been formulated to describe interacting systems that involve either one thermal transition (protein-ligand) or two thermal transitions (protein-protein) and either 1:1 or higher binding stoichiometry. Methods are described for obtaining binding constants and heats of binding by two different methods: calculation or simulation fitting of data. Extensive DSC data on 2'CMP binding to RNase are presented and analyzed by the two methods. It is found that the methods agree when binding sites are completely saturated, but substantial errors arise in the calculation method when site saturation is incomplete and the transition of liganded molecules overlaps that of unliganded molecules. This arises primarily from an inability to determine TM (i.e., the temperature where concentrations of folded and unfolded protein are equal) under weak-binding conditions. Results from simulation show that the binding constants and heats of binding from the DSC method agree quantitatively with corresponding estimates obtained from equilibrium methods when extrapolated to the same temperature. It was also found from the DSC data that the binding constant decreases with increasing concentration of ligand, which might arise from nonideality effects associated with dimerization of 2'CMP. Simulations show that the DSC method is capable of estimating binding constants for ultratight interactions up to perhaps 10(40) M-1 or higher, while most equilibrium methods fail well below 10(10) M-1. DSC data from the literature on a number of interacting systems (trypsin-soybean trypsin inhibitor, trypsin-ovomucoid, trypsin-pancreatic trypsin inhibitor, chymotrypsin-subtilisin inhibitor, subtilisin BPN-subtilisin inhibitor, RNase S protein-RNase S peptide, avidin-biotin, ovotransferrin-Fe3+, superoxide dismutase-Zn2+, alkaline phosphatase-Zn2+, and assembly of regulatory and catalytic subunits of aspartate transcarbamoylase) were analyzed by simulation fitting or by calculation. Apparent single-site binding constants ranged from ca. 10(5) to 10(20) M-1, while the interaction constant for assembly of aspartate transcarbamoylase was estimated as 10(37) in molarity units. For most of these systems, the DSC interaction constants compared favorably with other literature estimates, for some it did not for reasons unknown, while for still others this represented the first estimate. Simulations show that for proteins having two binding sites for the same ligand within a single cooperative unit, ligand rearrangement will occur spontaneously during a DSC scan as the transition temperature of the unliganded protein is approached.(ABSTRACT TRUNCATED AT 400 WORDS)
Article
The translational diffusion coefficient, D, of bovine serum albumin (BSA) is measured in a pH range from 7.0 to 2.0 as independent functions of ionic strength and BSA concentration. At relatively high ionic strength the diffusion coefficients are BSA concentration independent at relatively low concentrations and the molecule appears to expand (smaller diffusion constant) as the pH is lowered below the isoelectric point near pH 5. At low ionic strengths D increases as the pH is lowered and D is strongly dependent on the solution concentration, increasing linearly with concentration. However, the infinite dilution diffusion coefficient is ionic strength independent and indicates the same expansion as the pH is lowered from 5.0 to 2.0 as observed at high ionic strengths. These results are interpreted in terms of solution electrostatic effects.
Article
Studies on Sprague-Dawle rats have shown that the high-affinity corticosteroid-binding activity of serum increases after adrenalectomy or hypophysectromy. This increa e becomes apparent etween the first and sec nd day after adrenalectomy, goes up to approximately twice the normal binding activity and remains at this level for at least 3 weeks. Injection of large doses of corticosterone into the adrenalectomized rat decreases the corticosteroid-binding activity to the same low level as observed in n rmal rat after injection of the same dose of corticosterone. A species-specificity was demonstrated: the binding protein in rat ser m was found to bind corticosterone more firmly than cortisol, whereas the opposite relationship was observe for human transcortin. Electrophoretic studies showed the corticosteroid-binding protein in rat serum to be an alpha globulin. (Author)
Article
Small alkanes bind to β-lactoglobulin, ferrihemoglobin, and ferrimyoglobin, in each case, to a localized, interior, hydrophobic site with high affinity and nontrivial stereospecificity. In order to understand this behavior, the thermodynamic parameters for the transfer of pentane, neopentane, and butane to water, from ideal solutions, dodecyl sulfate micelles, ferrimyoglobin, ferrihemoglobin, and β-lactoglobulin, between 0 and 40° or 50°, were determined. ΔCp for the proteins, and for all solutes, is comparable with the dodecyl sulfate values; this variation of ΔH with temperature derives from the unusual behavior of water, and is typical of "hydrophobic" interactions. The heat of dissociation of butane and pentane from β-lactoglobulin and ferrihemoglobin is 3-4 kcal higher than from dodecyl sulfate micelles over the whole temperature range, and is the source of the high alkane affinity of these proteins. For neopentane, ΔH for β-lactoglobulin is more negative, and for ferrihemoglobin more positive, by 1-2 kcal, than the dodecyl sulfate values: β-lactoglobulin makes a clear distinction between pentane and neopentane, while ferrihemoglobin does not. The excess ΔH cannot arise from the solute-water interaction, from a nonoccurring displacement of water, or from exceptionally strong-solute-protein interactions, but must come from suboptimal interactions in the unoccupied site (plausibly, abnormally large molar and "free" volumes), which are normalized by strictly local rearrangements in the butane and pentane, but not (for β-lactoglobulin) the neopentane, complexes.
Article
Low-angle X-ray scattering, diffusion coefficients, and intrinsic viscosities have been calculated for several models of bovine serum albumin at low pH. These models are composed of spherical subunits, and include trimers, tetramers, and monomer-dimer mixtures in which the monomer is trimeric. Good agreement with experiment at pH 3.6 is obtained if a linear trimer model is used, in which the radius of the central sphere is 26.6 A, that of the two flanking spheres is 19.0 A, and adjacent subunits are touching. These results suggest that no substantial change in over-all dimensions of the protein has occurred by pH 3.6. At lower pH, expansion is considerable.
Article
The binding of anilinonaphthalenesulfonate by bovine serum albumin at various values of pH, temperature, and ionic strength has been studied by a fluorometric technique. Five moles of anilinonaphthalenesulfonate was bound/mole of serum albumin over the pH range 10-5.0. The equilibrium concentrations of free ligand, free protein, and complex were determined for values of the probability of binding from 0.05 to 0.95. The more complete titration curves obtained contained some 20 binary units of information. Abrupt changes in the apparent reaction order, similar to those demonstrated in the equilibrium of reduced diphosphopyridine nucleotide (DPNH) and beef muscle lactate dehydrogenase, were observed at pH 7, and a continuous increase in the reaction order with probability of binding was seen at pH 5. The results of pH 7, and probably also those at pH 5.0, are not expressible by an equation of Adair's type. We believe that, as in the case of lactate dehydrogenase, the results indicate the presence in the binding system of relaxation effects that are slow in comparison with the rate of dissociation of the ligand.
Article
The number of observations of both stable and transient partially folded intermediates is now sufficient for us to assume that all proteins, under the appropriate conditions, will form such species. These intermediates are characterized by substantial secondary structure and little tertiary structure; a collapsed conformation more compact than the unfolded state; exposed hydrophobic surfaces, which lead to binding of hydrophobic dyes and a propensity to aggregate; and a heat capacity similar to that of the unfolded state. The term compact intermediates encompasses a broad range of conformations and degrees of folding and compactness: compact intermediates have no single, unique conformation, but rather a whole plethora of structures that range from being very similar to the native state to being substantially expanded and significantly unfolded. The properties of compact intermediates from different proteins, and in some cases from the same protein under different conditions, may be significantly different. Equilibrium compact intermediates may be good models for transient intermediates formed during folding.
Article
HYDRO is a program for the calculation of sedimentation and diffusion coefficients, rotational relaxation times, and intrinsic viscosities of rigid macromolecules of arbitrary shape that are represented by bead models. Actually, HYDRO contains various FORTRAN callable subroutines that can be linked to the user's own programs to account for variability of shape or flexibility. Some hints are given for the use of HYDRO in various situations.
Article
Interaction with 8-anilino-1-naphthalenesulfonate (ANS) is widely used to detect molten globule states of proteins. We have found that even with stable partially folded states, the development of the fluorescence enhancements resulting from such interactions can be relatively slow and kinetically complex. This is probably because initial binding of the dye can induce subsequent changes in the protein structure, so that the ultimate resulting fluorescence enhancement is not necessarily a good, nonperturbing probe of the preexisting state of the protein. When ANS is used to study folding mechanisms the problem is compounded by the difficulty of distinguishing effects due to the development of dye interactions from those due to the changing populations of folding intermediates.
Article
The enhancement of the fluorescence quantum yield of 1,8-anilinonaphthalenesulfonic acid (ANS) upon binding to intestinal fatty acid protein (I-FABP) was exploited to devise an assay for free I-FABP. With this assay, we monitored the competition for free I-FABP between ANS and fatty acids and thereby extracted values for the dissociation constants (K(FA)) of fatty acids for I-FABP. We obtained these constants for the I-FABP ligands oleic acid, arachidonic acid, and palmitic acid. In addition, we measured the dependence of K(FA) for oleic acid upon temperature and at two pH values. From these data, we calculate the van't Hoff enthalpy of oleic acid binding. This enthalpy is compared with the enthalpies of binding obtained directly from titration calorimetry. Our experiments with the fluorescence-based assay generate values of K(FA) which disagree with older values obtained from calorimetry and other methods. Our own calorimetric data were analyzed with a view to improving the technique involved in subtraction of a "reference" dilution of the ligand into solution in the absence of the protein. By this maneuver, we obtained "corrected" titrations which could be fitted to values of K(FA) more in agreement with the values we determined via the fluorescence-based assay than wer the older literature values. Our new values for K(FA) also agree substantially with values derived using a complementary assay technique, one measuring the concentration of free fatty acid, that has recently been developed by Richiere et al [Richiere et al. (1995) J. Biol. Chem. 270, 15076-15084]. We compare the values of delta H degrees, delta S degrees, and delta C(p)degrees for fatty acid binding we have obtained in this work with those we found in earlier work with ANS binding to I-FABP [Kirk et al. (1996) Biophys. J. 70, 69-83]. Our interpretation of the origin of the thermodynamic changes for ANS binding in our earlier work is here substantiated and extended to include an evaluation in physical terms of the interaction of I-FABP with fatty acids.
Article
1-Sulfonato-8-(1')anilinonaphthalene (1,8-ANS) was employed as a fluorescent probe of the fatty acid binding site of recombinant rat intestinal fatty acid binding protein (1-FABP). The enhancement of fluorescence upon binding allowed direct determination of binding affinity by fluorescence titration experiments, and measurement of the effects on that affinity of temperature, pH, and ionic strength. Solvent isotope effects were also determined. These data were compared to results from isothermal titration calorimetry. We obtained values for the enthalpy and entropy of this interaction at a variety of temperatures, and hence determined the change in heat capacity of the system consequent upon binding. The ANS-1-FABP is enthalpically driven; above approximately 14 degrees C it is entropically opposed, but below this temperature the entropy makes a positive contribution to the binding. The changes we observe in both enthalpy and entropy of binding with temperature can be derived from the change in heat capacity upon binding by integration, which demonstrates the internal consistency of our results. Bound ANS is displaced by fatty acids and can itself displace fatty acids bound to I-FABP. The binding site for ANS appears to be inside the solvent-containing cavity observed in the x-ray crystal structure, the same cavity occupied by fatty acid. From the fluorescence spectrum and from an inversion of the Debye-Hueckel formula for the activity coefficients as a function of added salt, we inferred that this cavity is fairly polar in character, which is in keeping with inferences drawn from the x-ray structure. The binding affinity of ANS is considered to be a consequence of both electrostatic and conditional hydrophobic effects. We speculate that the observed change in heat capacity is produced mainly by the displacement of strongly hydrogen-bonded waters from the protein cavity.
Article
Matrix ligands are agents for isolating proteins out of dilute crudes by coprecipitating proteins. The ligands have a strong anion sulfonate head which initiates binding to proteins having a positive net charge, ZH+ approximately 5-20. Initial binding tightens protein conformation and starts to squeeze water from conformationally motile proteins. The tails are stackable hydrophobic organic groups, azoaromatic dyes which draw protein-ligand complexes together. Proteins coprecipitate as guests, in the ligand host matrix. In addition to stacking, ligand tails displace water because of their bulk, and lower the average dielectric constant near charged groups, which reinforces the electrostatic component of binding. Matrix ligands protect proteins, scavenge them from dilute crudes (0.01-0.1 per cent protein), and densify coprecipitates. Detergent ions in low concentrations, 10(-4)-10(-5) M also sometimes serve as coprecipitating agents, entangling their tails but probably not stacking. Divalent metal ions, Zn++, sometimes are useful auxiliary agents. Preparative scaleability from crudes is demonstrated starting from 100-200 g of raw peanuts and raw pineapple to coprecipitate a lectin and bromelain enzyme respectively in 1-2 h with 80-90 per cent activity yields. Ligands are released from coprecipitates by shifting the pH and trapping the ligands with exchange resins. Protein conformation tightening in solution is seen by viscosity measurements.
Article
The effect of pH, temperature, and partial pressure on the solubility of butane and pentane in solutions of bovine serum albumin has been studied. The binding of alkanes to bovine serum albumin is very sensitive to the conformation of the protein. The so-called F form, produced, in 0.15 M NaCl, in a single step around pH 4.1 marked by sharp but small changes in [α] and [η], binds only one-fourth as much butane and one-fifth as much pentane as native bovine serum albumin. In perchlorate solutions, the solubility reduction, like the changes in [a], occurs in two stages. The temperature dependence of the solubility indicates that butane is bound directly to some of the hydrophobic regions of bovine serum albumin, and the pressure dependence indicates that these regions are large. A variety of considerations leads to the conclusion that the binding sites are inside the bovine serum albumin molecule (i.e., most, if not all, the apolar side chains of these regions are not in contact with solvent). These data support Foster's model of bovine serum albumin: these regions, formed by the interaction between the hydrophobic surfaces of several substructures, would be disrupted when these substructures, without much internal rearrangement, separate to make the F form.
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