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Water-Insoluble Derivatives of Enzymes, Antigens, and Antibodies

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... This pioneering work was followed by a series of innovative studies which included preparation of insoluble papain and its use in studies on the structure of 7-globulin (Cebra et al., 1961), coupling of trypsin to a charged polymer and investigation of the effect of the polyelectrolyte field on enzyme activity Levin et al., 1964), and preparation of a collodion-papain membrane-the first example of a synthetic membrane-enzyme conjugate (Goldman et al., 1965(Goldman et al., , 1968a, followed by a collodion-alkaline phosphatase membrane (Goldman et al., 1971b). The various developments in the field of immobilized enzymes in Katchalski's laboratory and in others have been thoroughly reviewed (Katchalski, 1962a(Katchalski, ,b, 1969(Katchalski, , 1972Silman and Katchalski, 1966;Goldstein and Katchalski, 1968;Goldman et al, 1971a;Katchalski et al, 1971a). ...
... Nevertheless, work on enzyme immobilization became an important topic in research from the 1960s on, reflected by international conferences, notably, the Enzyme Engineering Conferences by the Engineering Foundation, New York, starting in 1971(e.g., (Weetall and Royer 1976, 1980. Notably, Ephraim Katchalski was among the pioneering groups providing insight into fundamental aspects and phenomena, including molecular characterization of the immobilized enzymes and mass transfer and efficiency (see, e.g., Levin et al. 1964;(Silman and Katchalski 1966); Gabel et al. 1971). ...
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Enzymatic penicillin hydrolysis by penicillin amidase (also penicillin acylase, PA) represents a Landmark: the first industrially and economically highly important process using an immobilized biocatalyst. Resistance of infective bacteria to antibiotics had become a major topic of research and industrial activities. Solutions to this problem, the antibiotics resistance of infective microorganisms, required the search for new antibiotics, but also the development of derivatives, notably penicillin derivatives, that overcame resistance. An obvious route was to hydrolyse penicillin to 6-aminopenicillanic acid (6-APA), as a first step, for the introduction via chemical synthesis of various different side chains. Hydrolysis via chemical reaction sequences was tedious requiring large amounts of toxic chemicals, and they were cost intensive. Enzymatic hydrolysis using penicillin amidase represented a much more elegant route. The basis for such a solution was the development of techniques for enzyme immobilization, a highly difficult task with respect to industrial application. Two pioneer groups started to develop solutions to this problem in the late 1960s and 1970s: that of Günter Schmidt-Kastner at Bayer AG (Germany) and that of Malcolm Lilly of Imperial College London. Here, one example of this development, that at Bayer, will be presented in more detail since it illustrates well the achievement of a solution to the problems of industrial application of enzymatic processes, notably development of an immobilization method for penicillin amidase suitable for scale up to application in industrial reactors under economic conditions. A range of bottlenecks and technical problems of large-scale application had to be overcome. Data giving an inside view of this pioneer achievement in the early phase of the new field of biocatalysis are presented. The development finally resulted in a highly innovative and commercially important enzymatic process to produce 6-APA that created a new antibiotics industry and that opened the way for the establishment of over 100 industrial processes with immobilized biocatalysts worldwide today.
... The results were published [33,34], as well as monographs collecting most of the relevant research results both by academic and industrial research (e.g., [35]). Among the pioneer groups providing insight into fundamental aspects and phenomena, including mass transfer and efficiency, was that of Katchalski (see e.g., [36]). ...
Applied biocatalysis has its roots in ancient Mesopotamia, China, and Japan, in the manufacture of food and alcoholic drinks. The history of enzyme technology is, of course, an essential part of the history of biotechnology. First scientific observations on enzyme activities in the late eighteenth century were by Spallanzani and Scheele. A remarkable process was developed by Schutzenbach in 1823: an acetic acid fermentation process known as "fast acetic acid manufacture". From about 1894 onwards, Emil Fischer investigated in a series of experiments the action of different enzymes using several glycosides and oligosaccharides; the results revealed specificity as one of the key characteristics of enzymes. In his first paper on alcoholic fermentation without yeast cells (1897), Buchner described, in a remarkably short and precise manner, the separation of the (alcoholic) fermentation from the living yeast cells by an extract. Fischer in his work elaborated the essential aspects of enzyme catalysis during the 1890s.
... Moreover, these immobilized REVIEW trypsin preparations showed excellent digestion reproducibility based on liquid chromatographic and capillary electrophoretic peptide maps. Insoluble trypsin preparations were found to be considerably more stable than native trypsin in the alkaline pH range (79). Glassmeyer and Ogle (80) reported that an insoluble trypsin preparation could be used repeatedly without loss of activity and could be left standing in pH 8.0 buffer at room temperature for 40 h with only a 9% loss of activity. ...
Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.
... The immobilization of enzymes was initially done by the use of polymer-based matrices and linkage onto carrier materials. Considerable efforts have been made for crosslinking of enzymes either using protein-based methods or using inactive materials (Silman and Katchalski 1966). The history of confining enzymes is dated back to the 1950s. ...
Free enzymes do not possess properties of recovery and reusability, and also they are not stable at wide pH and temperature range. Therefore, new ways which can enhance enzyme stability and reusability should be developed, and hence, the immobilization technique is one such approach. These immobilization techniques offer such materials which have the ability to be active in the much wide range of pH and temperature, and also they are more stable than the free enzymes. Immobilization is carried out on the nanosized material either by adsorption, covalent coupling, entrapment, encapsulation or cross-linking. These nanomaterial-immobilized enzymes show several advances over the free enzymes because of large surface area-to-volume ratio, lower mass transfer resistance and high mobility. Several nanomaterials are used for immobilizing the enzymes; however, their recovery from the reaction mixture is very poor. Therefore, the magnetic nanomaterials are more attractively used in immobilization because the enzyme immobilized through magnetic nanomaterial has the tendency to be easily separated out from the reaction mixture. These nanomaterial-immobilized enzymes show wide range of applications in biotechnology, bioanalysis, biomedicine, pathology and biosensors.
... Another study by Glassmeyer and Ogle, who used water to store the product at 4 °C, concluded that there was a maximum of a 15% drop in the activity in the trypsin after several months 65 . In 1966, Silman and Katchalski said after the lyophilization experiment that they could not find any notable decrease in the activity when it was stored at room temperature 66 . ...
... Recently, the immobilization of enzymes has been the subject of increased interest, and potential applications of these enzyme derivatives have been published (4,10). In previous papers (5,6,9,11,12), we reported on the immobilization of aminoacylase, aspartase, and asparaginase. ...
The immobilization of asparatase-containing Escherichia coli was investigated by various methods, and the most active immobilized cells were obtained by entrapment in a polyacrylamide gel lattice. Other asparatase-containing bacteria were also entrapped by the same method, and the enzymatically active immobilized cells were obtained. The aspartase activity of the immobilized E. coli cells was increased nine- to tenfold by autolysis of the cells entrapped in the gel lattice. Enzymatic properties of the immobilized E. coli cells were investigated and compared with those of the intact cells. The optimal pH was 8.5 for the immobilized cells and 10.5 for the intact cells. The aspartase activities of immobilized and intact cells were not activated by Mn²⁺, which can activate the immobilized and native aspartases. The heat stability of the immobilized cells was somewhat higher than that of the intact cells. Bivalent metal ions such as Mn²⁺, Mg²⁺, Ca²⁺ protected against thermal inactivation of the aspartase activity of the immobilized and intact cells.
Points of contact between chemical engineering and biology are discussed in an attempt to see what the older concepts of chemical engineering may contribute to the understanding and mathematical analysis of various biological operations with or without membranes. The appendix gives a mathematical treatment of a diffusion and reaction problem in enzyme kinetics.
This chapter provides an overview of membrane phosphohydrolase. The phosphohydrolases are a group of enzymes that have been studied most extensively from the viewpoint of their association with membranes. ATPase is regarded as a cell membrane component concerned with electrolyte transport; glucose-6-phosphatase is a cell endoplasmic reticulum hydrolase and transferase; 5′-nucleotidase is associated with brush border cell membranes; acid phenylphosphatase is a mouse kidney endoplasmic reticulum and lysosomal enzyme; and alkaline phosphatase is a brush border cell membrane component. Acid phosphatase is widely considered to be exclusively a lysosomal enzyme. In this chapter, evidence is presented that indicates that acid phosphatase is a component of membranes in epithelial cells of mouse kidney and that lysosomal and microsomal acid phosphatases are two different isoenzymes. The chapter further discusses properties that different membrane phosphohydrolases may have in common.
Currently, there is an upsurge of interest in the immobilisation and regimentation of biologically active molecules, notably enzymes, anti-bodies and various bio-specific inhibitors, by conjugation with synthetic and semi-synthetic carriers. The resulting materials are of practical importance because they are recovered readily from heterogeneous reaction mixtures by filtration and, in certain cases, may be used continuously in perfused or fluidised beds. More important from a theoretical viewpoint, choice of a suitable carrier for the immobilisation process enables the researcher to define precisely the microenvironment of the constrained biological molecule. Simulation of the native, cellular environment of the molecule and, further, the creation of new, artificial environments, calculated to modify and perhaps improve, biological activity become distinct possibilities.
Immunochemical Potentiometric and amperometric electrodes are in the infant stages of development, yet the literature is already replete with diversified efforts to design increasingly sensitive and specific immunoelectrodes (Eggers et al., 1982; Guilbault, 1983; Boitieux et al., 1984; Keating and Rechnitz, 1984). The carbon dioxide gas-sensing probe, however, has played only a limited role in this research. Despite all the schemes designed for immunosensors, there is a conspicuous absence in the use of the carbon dioxide probe. Why this might be so is suggested by the constraints within which use of the carbon dioxide probe is practical as well as the limiting characteristics of the immunochemical reaction being studied. Additional restrictions result from the presence of the enzyme when developing an enzyme immunoassay [EIA]. Despite this, the enzyme is a favorite “transducer”, or label, which produces a signal related to the immunochemical reaction and can be recognized by the electrochemical detector. This integration of enzyme chemistry, immunochemistry and probe characteristics is a critical factor in determining, during method development, the feasibility of selecting a carbon dioxide probe as the analytical detector. These key ingredients will be discussed in this overview together with possibilities for the future of the carbon dioxide sensor in immunochemical applications. Improvements in the probe and broader applications will highlight this future.
It is well known that the immobilization may alter both the chemical and physical properties of an’enzyme, such as its pH-activity behavior, apparent saturation constant, Km, substrate specificity and stability toward conformational inactivation, in addition to simply restricting its gross physical movement. The enzyme activity is generally decreased through immobilization. It is necessary for obtaining an immobilized enzyme preparation of high activity to discuss the processes of enzyme immobilization and investigated the factors affecting the activity and yield of immobilized enzyme. In the present work, we discussed the processes of enzyme immobilization by microencapsulation and clarified the factors affecting the activity and yield of enzyme immobilized.
chromatographic separation;gas-solid column;chromatographic column;molecular interactions;gas liquid chromatography
Immobilized enzymes are used in organic syntheses to fully exploit the technical and economical advantages of biocatalysts based on isolated enzymes. Immobilization enables the separation of the enzyme catalyst easily from the reaction mixture, and can lower the costs of enzymes dramatically. This is true for immobilized enzyme preparations that provide a well-balanced overall performance, based on reasonable immobilization yields, low mass transfer limitations, and high operational stability. There are many methods available for immobilization which span from binding on prefabricated carrier materials to incorporation into in situ prepared carriers. Operative binding forces vary between weak multiple adsorptive interactions and single attachments through strong covalent binding. Which of the methods is the most appropriate is usually a matter of the desired applications. It is therefore the intention of this paper to outline the common immobilization methods and reaction technologies to facilitate proper applications of immobilized enzymes.
Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost-effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co-immobilized them on modified poly-p-hydroxybutyrate synthase (PhaC)-inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled-coil interaction. Enzyme-loaded particles could be easily purified by centrifugation. Total conversion of 4'-chloroacetophenone to (S)-4-chloro-α-methylbenzyl alcohol could be accomplished using enzyme-loaded particles, catalytic amounts of NAD(+) and formate as substrates for FDH. Chiral GC-MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost-effective alternative to coupled reactions using purified enzymes.
Over the years, the science of biosensors has evolved significantly. The first or earliest generation of biosensors only detected either the decrease or increase of product or reactant-based natural mediators as the pathway for electron transfer. The subsequent second-generation biosensors were biomolecule based and used artificial redox mediators, such as organic dyes to detect and to increase the reproducibility and sensitivity of the result. However, the recent generation of biosensors work mostly on the principle of electron mobility, with different criteria, such as selectivity, precision, sensitivity, etc., can be used to quantify, efficiently. This review deals with exploring the scope and applications of Immobilized lipase biosensors. Generally, Triglycerides or TG molecules are either detected using Gas Chromatography or, using a chemical or an enzymatic assay. Immobilization of lipase on solid supports has led to increased stability and reusability of the enzyme in non-aqueous solvents. With better enzyme performance, efficient product recovery, and separation from the reaction, immobilized lipase biosensors are garnering increasing interest worldwide. Along with so many advantages including but not limiting to ones mentioned earlier, immobilized lipase-based biosensors come with their own set of challenges, such as the partitioning of the analyte with aqueous medium, slower reaction rate, etc., they have been discussed in the following review. Alongside, we also review the development of a new generation of biosensors and bioelectronic devices based on nanotechnology.
In recent years, the importance and application of entrapped whole cells have been so frequently proposed, discussed, put into practice, and reviewed (1–3) as to need no further reemphasis here.
Enzymes are employed in several fields of basic and applied research as biocatalysts in green chemistry, biosensor, nanobioelectronics, biofuel, and pharmaceutical, agricultural, and biotechnological industries. In present scenario, the diminution of fossil fuels gained the attention of researchers for substitute and sustainable renewable energy resources for biofuel production to combat worldwide energy consumption. The enzyme immobilization as biocatalysts for biofuel applications from lignocellulosic biomasses is found to produce highest percentage of bioethanol. The enzyme immobilization is a fundamental tool to reduce the cost and harness their benefits. The stabilization of enzymes using immobilization helps in efficient recovery from the reaction conditions after biocatalysis and hence makes laborious separation steps easy and permits repetitive use of enzymes. Besides this, it offers several other advantages such as stabilization against harsh reaction conditions, thermodynamic and kinetic stability, surface- and volume-confined enzyme environments, ability to design multi-step reaction, and reduced formation of undesired products which makes easy separation of soluble end products than free enzymes. The different methods of enzyme immobilization either involve adsorption or covalent bonding or encapsulation or a combination of different methods. Several types of nanoparticles and nanocomposites are being used for the stabilization of enzymes which retain the enzyme activity even after immobilization. This book chapter will cover the developments in coupled strategies and the deeper knowledge in stabilization of enzymes with special emphasis on the possibilities of nanomaterial coupled immobilization for operational stabilities in biofuel application.
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Leucine aminopeptidase and aminopeptidase M have been covalently bound to an arylamine derivative of porous glass. The bound forms of both enzymes retain 100% of their activities at saturating levels of substrate (leucine p-nitroanilide). For the hydrolysis at pH 7.3 and 25° κcat values for bound and free leucine aminopeptidase are 46 ± 5 s⁻¹ and 46 ± 2 s⁻¹, respectively; for aminopeptidase M (pH 7.5 and 25°) κcat values are 23 ± 2 s⁻¹ and 21 ± 0.4 s⁻¹, respectively. Although the Michaelis constants for both enzymes increase on binding, the pH and temperature dependencies of the bound enzymes remain unchanged. These data suggest that the environments and conformations of the enzymes are not significantly changed after coupling to the solid support. The apparent decrease in the binding of substrate could be explained by a decrease in the effective diffusion coefficient of the substrate. Both insoluble enzymes are active against polypeptide substrates. After treatment for removal of contaminating endopeptidases, the immobilized derivatives of leucine aminopeptidase and aminopeptidase M were used successfully in NH2-terminal sequence determination. The bound aminopeptidase M appears to be the better of the two for this purpose. Both bound enzymes will catalyze the hydrolysis of the aminoethylated A and B chains of insulin nearly to completion (≥87% recovery of free amino acids in all cases). These digests are carried out at pH values near neutrality in a volatile buffer with no activating metal. Immobilized pronase (Royer, G. P., and Green, G. M. (1971) Biochem. Biophys. Res. Commun. 44, 426) was used in concert with bound leucine aminopeptidase and bound aminopeptidase M for the hydrolysis of β-lactoglobulin. In both cases the recovery of free amino acids was 93%. These bound enzymes should be quite useful in amino acid composition determinations when acid-labile residues such as tryptophan, glutamine, asparagine, or certain “affinity labeled” side chains are present.
This chapter discusses the control of the Krebs cycle. The Krebs cycle plays a key role in aerobic respiration. It was formulated by H. A. Krebs and W. A. Johnson when they described the formation of citrate from oxaloacetate and pyruvate. This condensation reaction linked the series of reactions into a cycle, which in its current version is usually called the tricarboxylic acid (TCA) cycle. While considering the control of the TCA cycle, there are a number of possible regulatory sites that must be recognized. The cycle is dependent on an input of carbon, usually in the form of acetyl-CoA, which can be derived from carbohydrate via glycolysis or from fat via β-oxidation. However, in plants, the operation of a glyoxylate cycle converts the acetyl-CoA derived from fatty acids to succinate. Amino acids and other organic acids can also feed into the cycle. Substrate control can be imposed either by restricting the supply of substrate or by restricting the rate of entry of the substrate. Control can also be applied on the enzymes involved in the TCA cycle; however, a restriction imposed on one enzyme does not apply to the entire process unless it is operating as a complete cycle.
Mobile phones finds wide application in audio video and image communication .We can easily transfer multimedia data from one mobile phone to another in few seconds. The present work highlights an innovative approach of FMS to transfer fragrance from one mobile to another. The proposed system will be having a fragrance assembly in mobile with a control and transfer signal method from sender to receiver.
Affinity purification followed by mass spectrometry has become the technique of choice to identify binding partners in biochemical complexes isolated from a physiologic cellular context. In this report we detail our protocol for tandem affinity purification (TAP) primarily based on the use of the FLAG and HA peptide epitopes, with a particular emphasis on factors affecting yield and specificity, as well as steps to implement an automated version of the TAP procedure. © 2019 by John Wiley & Sons, Inc.
Realization of the potential of immobilized enzymes as a new type of model system for the investigation of isolated aspects of complex biological phenomena on the one hand, and of their industrial potential as a new type of highly specific heterogeneous catalyst for continuous processes on the other, brought together chemical engineers, organic and physical chemists, biochemists, biologists, and microbiologists. Derivatized polymers with groups of different chemical specificities are needed for attaining biologically active immobilized preparations of different proteins. The chemical nature of the support material might determine not only the amount of bound protein, but also the extent to which its biological activity is retained. Protection of the enzyme by chemical modification prior to coupling might be necessary. Immobilized derivatives of trypsin and chymotrypsin were used for the selective adsorption of the pancreatic inhibitors of these enzymes from crude extracts. The inhibitors were eluted under conditions where binding was weakest. The reversal of the procedure, that is, the use of the purified inhibitors in immobilized form for the isolation of pure enzymes, was a natural extension of the same basic concept.
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Anti-enzyme antibodies can be utilized for various purposes. They have been used for single step immunoaffinity purification of enzymes and for their immobilization. Both polyclonal and monoclonal antibodies have been employed in the immobilization of enzymes. The resulting immobilized enzymes exhibit almost full activity and enhanced stability. Antibodies raised against the peptides corresponding to the labile region of enzymes can be used to prepare affinity supports that bind and selectively confer enhanced stability to them. Antibodies can facilitate folding and prevent aggregation of protein antigens. This chaperone-like antibody activity may prove to be a promising approach to the treatment of Alzheimer's and prion-related diseases. The antibodies have immense therapeutic potential and are being utilized to treat various diseases. This review aims at giving an overview on the stabilization of enzymes by antibodies and the therapeutic potential of the anti-enzyme antibodies.
The term affinity chromatography seems to have been first used by Cuatrecasas et al. (1968); however, the principles involved have long been known in the field of immunology. In fact, as early as 1936, Landsteiner and van der Scheer used insolubilized haptens for the isolation of the corresponding antibodies. Since then numerous examples of antibody purification by use of immunosorbents have been reported (Silman and Katchalski, 1966).
Mit der Entstehung des Lebens auf der Erde ist die Bildung der Enzyme untrennbar verknüpft. Der gesamte in der Zelle und damit in der lebenden Natur sich vollziehende Stoffwechsel wird durch Enzyme gesteuert, von denen z. Z. etwa 2000 bekannt sind. Es wird angenommen, daß die Anzahl der in einer Zelle insgesamt wirksamen verschiedenen Enzyme zwischen 2000 und 10000 liegt.
Polymer blends, grafts, and blocks have achieved importance as toughened plastics and novel elastomers because of their complexity, not in spite of it. By definition, these materials are characterized as some intimate combination of two (or more) kinds of polymer molecules. Because of the very small entropy gain on mixing long polymer chains, and the usually encountered positive heat of mixing, most polymer blends, grafts, and blocks form two phases. Most strikingly in these materials, the exact mode of synthesis controls the two-phase morphological features, which in turn influences their mechanical behavior. Thus a one-to-one relationship exists between synthetic detail and potential application.
In recent years much attention has been devoted to the techniques of affinity chromatography for studying complex biological systems1. The basic principle is to immobilise one of the components of the interacting system by binding to an insoluble support, preferably through covalent bonds2,3. The biologically active support can be used to separate selectively from the reaction mixture the component with which it interacts. A successive elution yields pure components4–6. This technique consequently looks very promising both as an analytical method7 and for studying the mode of action of biological substances8,9.
This chapter presents a synthetic approach to the study of microenvironmental effects on enzyme action. The chapter discusses water-insoluble enzyme derivatives. Enzyme derivatives in which the biologically active protein is covalently bound to a water-insoluble polymeric carrier may serve as easily removable reagents of considerably improved stability; they are well suited for repeated or continuous use and provide means for a more adequate control of enzyme reactions. Immobilized enzyme derivatives can be used as specific adsorbents for the isolation and purification of enzyme inhibitors. Moreover, new properties may sometimes be imposed on the immobilized enzyme by the chemical nature of the polymeric carrier. In the case of acidic proteins, containing large excess of carboxyl groups, immobilization could be achieved by coupling the protein to aminoethyl cellulose through soluble carbodiimide activation of the protein carboxyls. The kinetic behavior of immobilized enzyme systems is dominated by several factors not encountered in the kinetics of free enzymes: (1) effects of the chemical nature of the carrier, stemming from the modified environment within which the immobilized enzyme is located, (2) steric restrictions imposed by the carrier, and (3) diffusion control on the rate of substrate penetration.
Ephraim Katchalski-Katzir was a leading researcher in the development of polyamino acids, which allowed new insights into the reactions and behaviour of proteins. His work developed pathways into the exploration and investigation of antigenicity. He was a founder of the Israel Society for Biochemistry and a founding member of the European Molecular Biology Organization. He was also president of Israel between 1973 and 1978—an unusual distinction because this post was usually held by former politicians. As head of the Israeli Association of Science, he was prominent in promoting the cause of science both as president and outside his term of office. Ephraim Katchalski-Katzir passed away in May 2009.
This chapter discusses chemical modifications of bovine trypsinogen and trypsin. The present knowledge of trypsin has closely paralleled analogous studies of bovine chymotrypsin, an enzyme with similar catalytic characteristics but with different enzymatic specificity. In fact, the functional similarities between trypsin and chymotrypsin and their homology of primary structure have led to proposals of their common origin by gene duplication and divergence from an ancestral protein. The events leading to the functional aspartyl-histidyl-seryl interaction comprise the phenomenon of trypsinogen activation. The activation process is initiated by specific cleavage of the Lys6-Ile7 bond to remove the highly charged hexapeptide (Val-Asp4-Lys) from the N-terminus leaving the hydrophobic sequence (Ile-Val-Gly-) with a charged, α-amine function. However, a conformational change accompanies the appearance of activity, and it has never been demonstrated whether the explicit molecular event creating activity is the appearance of the new α-amino group, the loss of the hexapeptide, or the consequential refolding.
A nursery rhyme, which some of us learned when we were children brought tinker, tailor, soldier, and sailor together (Opie and Opie, 1951), rather like this volume, where its authors have been convened from many disciplines to describe how the diverse indexes of our individuality—our proteins—can be handled for biomedical purposes. My contribution to the mix is a chapter on strategies about enzyme replacement therapy; other authors provide the tactical details upon which the successful campaign for such treatment will depend.
It has been shown that the inhibitory capacity of antibody obtained in the course of immunization, first subcutaneously in adjuvant and then by repeated intravenous injections, varies in the course of immunization and that there is a progression towards completely inhibitory antibody. The degree of inhibition exercised for a given antibody varies with the sequence of molecular weights of products; thus the inhibitory capacity is greatest in the reaction of enzyme on nucleic acid, is smallest in the inhibition of the formation of 3′ cytidylic acid, and is intermediate with respect to the formation of cyclic dicytidylic acid. The effect of antibody is identical in the inhibition of two very different reactions, resultng in the formation of products of similar molecular weight, 3′ cytidylic acid and the methyl ester of 3′ cytidylic acid. The above observations are best explained by the assumption that the inhibitory capacity of antibody depends on a mechanism of steric hindrance.
The procedures for and applications of the hemagglutination and inhibition reactions with bis-diazotized benzidine-protein conjugated cells have been described. These include special features which influence their applicability, the relative sensitivity of the tannic acid, BDB, and Coombs' methods, and recently discovered general applications. The latter include the estimation of concentrations of proteins in complex mixtures in tissue extracts and serum and the precise measurement of proteins by conversion of hemagglutination to a hemolytic reaction. It was concluded that for most purposes the hemagglutination reaction with tannic acidprotein cells was preferable to that with BDB cells. The major exception was that of systems in which the tannic acid method yielded non-specifically agglutinable cells.
MODIFIED celluloses have been combined chemically with a variety of enzymes, with retention of enzymatic activity but with interesting and useful modifications of some properties. Derivatives have been prepared with carboxymethylcellulose and diazobenzylcellulose.
The BDB-hemagglutination technique has been adapted for the demonstration of antibodies to grass pollen constituents in the sera of immunized rabbits and grass-sensitive hay fever patients. The grasses used were timothy, June grass, orchard grass, redtop, sweet vernal, and Bermuda grass. The method was shown to be highly sensitive. Titers of the order of 104 and 102 were obtained with sera of immunized rabbits and of allergic human subjects, respectively. The specificity of the technique was demonstrated by inhibition of the hemagglutination with free soluble pollen constituents.The inhibition of hemagglutination obtained with rabbit antisera was used for establishing antigenic relationships among the various grasses. Timothy, June grass, orchard grass, redtop, and sweet vernal were found to contain common antigens, which were different from those of Bermuda grass and ragweed. The results of Prausnitz-Küstner tests using the cross-neutralization technique with desensitizing doses of each of the five grasses (timothy, June grass, orchard grass, redtop, and Bermuda grass) suggest that redtop grass pollen contains all the allergens common to the other grasses and that timothy, June grass, and orchard grass pollens have a complex and in many respects similar allergenic composition and contain additional allergens to those found in Bermuda grass pollen.Absorption of an allergic serum with adsorbents prepared by coupling grass pollen constituents to polystyrene or erythrocytes resulted in the removal of the hemagglutinating factor(s), which was paralleled by the removal of skin-sensitizing antibodies.
Immunoadsorbents with a large capacity for antibody adsorption were prepared by coupling antigens to insoluble polymers of rabbit serum albumin or γ-globulin. The insoluble polymers were made by coupling mercaptosuccinyl groups to protein and then crosslinking the modified protein with tris(1-(2-methyl)aziridinyl)phosphine oxide (MAPO). With an appropriate number of hapten groups incorporated, the adsorbents adsorbed more than eual their weight of anti-hapten antibody with high efficiency. The capacity of the adsorbent for anti-bovine serum albumin antibody was somewhat lower, but still much higher than that for adsorbents previously reported. That antibody content of the purified antibody preparations was higher than 90 per cent.
1.1. Antibody to subtilisin, which had been reacted with subtilisincoated cellulose, retained immunologically active sites for additional subtilisin binding.2.2. Carboxypeptidase passed through such an antigen-antibody column was freed of detectable amounts of subtilisin-like activity and was recovered almost quantitatively without loss of specific activity.
A general system for the purification of proteins utilizing the specificity of immunochemical reactions has been described. The system makes available highly purified antibody as a reagent without requiring dissociation from antigen. Data on the effectiveness of the method and discussion of its possible applications and limitations are presented.
THE isolation of precipitating antibodies in a purified form has been the subject of several investigations during the past few years. The property of the specific combination of antibodies with their homologous antigens has been exploited for this purpose. In principle, a general procedure for the isolation of antibodies would involve the following steps: (1) formation of insoluble antibody-antigen complexes, (2) dissociation of these complexes, and (3) separation of the purified antibodies from the antigen(s). Obviously, the last step would be facilitated if the antigen were insoluble or could be made insoluble by coupling it via stable covalent chemical bonds to a supporting medium. For example, in previous investigations protein antigens were coupled to fibrinogen1, ion-exchange resins2 and cellulose3. Since these supporting media were endowed with ion-exchange properties, it is conceivable that serum proteins may be bound to them non-specifically. In order to avoid or at least to minimize non-specific absorption of serum proteins, in the present study, protein antigens were coupled through stable azo bonds to a non-polar polystyrene (Dow `Styron PS-2' obtained through the courtesy of the Physical Laboratory, Dow Chemical Co., Midland, Michigan) framework. For this purpose polystyrene was nitrated4. The ensuing polynitro polystyrene was then reduced to polyamino polystyrene4,5 and the latter compound diazotized. The soluble protein antigens were then coupled to the polydiazotized polystyrene at pH 7.5. The resulting insoluble Styron-antigen conjugates are dark brown precipitates. At the beginning of the study we were not aware that a somewhat similar procedure had been used for coupling proteins to polystyrene6,7.
1. An allergenically active fraction was isolated from ragweed pollen which was 50 times as active, on a weight basis, as the dialyzed residue of the whole water soluble extract of ragweed pollen. 2. This material consisted of a straw colored pigment and peptide(s) composed of eight amino acids: lysine, arginine, glycine, glutamic acid, hydroxyproline, valine, alanine and norleucine. It did not contain any carbohydrate. 3. The allergenic activity was destroyed after hydrolysis with 6 N HCl at 100°C for 15 min, but was not diminished on standing in 6 N HCl at 25°C for 8 hr or if heated in boiling water at pH 7.0 for 14 hr. 4. The active material gave positive ring tests with both rabbit and goat antiragweed pollen sera. 5. The available evidence suggests that peptide(s) may account for all the allergens in ragweed pollen.
THE communication of Gyenes, Rose and Sehon1 prompts us to record briefly some observations in a closely related field.
Proteins coupled to polystyrene by azo linkages were used as solid adsorbents for the determination of antibodies to soluble proteins. Studies were carried out quantitatively using I131 labeled bovine serum albumin and ovalbumin and their antibodies. Antibodies specifically combined with antigen complexes could be eluted efficiently at pH 3 (citrate buffer) whereas antigens and nonspecifically adsorbed proteins remained firmly bound to the resin. Elution of antibodies took place slightly in 10% sodium salicylate and at 60°C, pH 8, but not in 17% NaCl. Eluted antibodies showed normal precipitin curves. By diluting anti-BSA with normal globulin, it was found that 0.2 μg antibody in 1 mg globulin could be detected. Five-tenths to two micrograms of antibody/mg globulin could be determined within 10% accuracy. The nature of the effective antigen in the polystyrene-protein complex is not well defined. The original aminopolystyrene or the diazopolystyrene quenched with β-naphthol bound antigen proteins very strongly but to a lesser extent than did the diazopolystyrene itself. β-Naphthol-quenched diazopolystyrene treated with BSA was tested and found to be almost as effective as the chemically coupled BSA in purifying antibodies.
The acetylation of trypsin with acetic anhydride results in a heterogenous mixture of acetylated derivatives. That fraction which is most fully acetylated (85–90%) is enzymically inactive, whereas the partially acetylated (about 50%) fraction is fully as active as unmodified trypsin. These two forms of trypsin may be further differentiated on the basis of their solubility properties, chromatographic behavior on DEAE-cellulose, and optical rotation. The implication of these results with respect to the structural requirements for enzymic activity is discussed.
Anti-Rp antibody can be separated into a number of fractions by adsorption on a specific immunoadsorbent followed by stepwise elution with increasing concentrations of benzenearsonate over the range of 10−6-10−1M. Under a given set of conditions, this method of fractionation yields an elution pattern that is distinctive for a given sample of antibody. Upon refractionation each fraction yields a different elution pattern reflecting the concentration of hapten which eluted the fraction previously. There is a very wide heterogeneity in relative binding constants of antibody for immunoadsorbents and hapten since some antibody requires 105 times as concentrated hapten for elution than other antibody. As the hapten concentration is increased, the binding constants of eluted antibodies for p-(p-hydroxyphenylazo)benzenearsonate and p-iodobenzenearsonate decrease; but the constants for the former decreases much more rapidly. These results are interpreted as indicating that the heterogeneity observed is due to variations in structure of the combining sites.
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