International Journal of Peptide and Protein Research

Online ISSN: 0367-8377
Publications
Article
Several synthetic routes are reported to prepare the hetero diprotected 1,1-diaminoalkanes from N-acylated amino acids or peptides for incorporation into partially modified retro-inverso peptides. The Curtius rearrangement was carried out on the N-protected aminoacyl azides obtained from the N-protected aminoacyl hydrazide by nitrosyl chloride or by sodium azide reaction with an appropriate mixed carboxylic carbonic acid anhydride. The resulting isocyanate was allowed to react with alcohol to give a urethane-type protecting group or, via a "one-pot" approach, directly with a carboxyl carrying component to yield the modified (reversed) peptide bond. The carboxyl component can be either an N-acylated amino acid or a malonic acid. The more standard route involves selective deprotection of the 1,1-diaminoalkane residue followed immediately by coupling with a carboxyl component to yield the same modified peptide derivative.
 
Article
1,1,4,4-Butanetetracarboxylic acid (BTCA) is evaluated as an analogue for the metal binding site in dipeptides of gamma-carboxyglutamic acid (Gla). Molecular modeling suggests that the four carboxylic acid groups in BTCA can assume a similar conformation to the four gamma-carboxylic acid groups in GlaGla and thus provides the impetus for the synthesis and metal binding determinations. BTCA is synthesized via the tert.-butyl ester and characterized via NMR, mass spectroscopy, and elemental composition. Equilibrium binding constants with protons, Ca(II) and Mg(II) are determined via pH and Ca(II) ion-selective electrode titrations and are found to be similar to those for GlaGla peptides with blocked termini.
 
Article
The reactivity of arginine residues in ovine prolactin was studied by reaction with 1,2-cyclohexanedione. Kinetic analysis of the data showed a good fit with two simultaneous pseudo-first-order equations with apparent velocity constants of 0.28 and 1.2 × 10−2 min−1, corresponding to 1.8 ‘fast’ and 8.7 ‘slow’ residues, respectively. Modification led to a decrease in binding capacity to lactogenic rat liver receptors, and apparently the modification of the two ‘fast’ reacting arginine residues is responsible for the rapid loss of this capacity. The presence of a non-reacting arginine has been described in human and bovine growth hormones, and it is located near the carboxy-terminus. This lack of reactivity is probably due to the formation of a salt bridge, since the arginine residue becomes susceptible to modification once the peptide is separated from the rest of the molecule. This salt bridge is absent in ovine prolactin, since the homologous arginine residue is reactive with cyclohexanedione. This result suggests that there could be a difference between the three-dimensional structure of ovine prolactin and of the growth hormones, at least near the carboxy-terminal region of the molecule.
 
Article
Deprotection of Nin-formyl tryptophan (Trp) occurs during liquid hydrogen fluoride treatment at 0 degrees when 1,2-ethanedithiol (EDT), or 1,4-butanedithiol, is present. Deformylation, as evidenced by amino acid analysis and ultraviolet spectral analysis, is complete after 10 min at 0 degrees when Trp(CHO) is treated with HF:anisole:EDT(85:10:5) or HF:EDT (95:5). HF treatment of a peptidyl-resin containing Trp(CHO) yielded a peptide whose ultraviolet spectrum was typical of Trp (maximum at 280 nm) rather than Trp(CHO) (maximum at 300 nm). However, in the absence of dithiol during HF treatment, the expected spectrum for Trp(CHO) was obtained. The efficiency of HF cleavage of a 49-peptidyl-resin was unaffected by EDT; 77% was cleaved in the presence of EDT, and 76% in the absence of EDT. In a model study, dithiol deformylation as a synthetic tactic was used for the solid-phase synthesis of Trp-Met-Asp-Phe amide. When the Trp(CHO)-Met-Asp(Bzl)-Phe-NH-methylbenzhydrylamine resin was treated with HF:anisole:EDT(85:10:5) for 30 min at 0 degree, the major peptide component observed by high pressure liquid chromatography (HPLC) was identical to the control tetrapeptide amide made without CHO-group protection of Trp.
 
Article
Arginine residues (5.5 out of 6) of the trypsin-kallikrein inhibitor from bovine organs (Kunitz inhibitor) were selectively modified by reaction with 1, 2-cyclo-hexanedione in sodium borate buffer, pH 9.0. The modified inhibitor is still highly active in inhibiting trypsin and chymotrypsin at 1:1 inhibitor: enzyme molar ratio and full inhibition was achieved at slightly higher molar ratio. The extent of correct refolding, upon reoxidation, of the reduced, arginine-modified inhibitor is diminished and regeneration of two arginines occurred under the reduction conditions. The stability constants and the standard-free energies of binding of the complexes between trypsin, or chymotrypsin, and the native, the arginine-modified and the reduced and reoxidized arginine-modified inhibitor have been determined from inhibitory assays.
 
Article
A new heterobifunctional cross-linking reagent, 1,2,3-thiadiazole-4-carboxylic acid, for the photochemical conjugation of peptides to proteins is described. The title compound can be coupled directly to a protected peptide resin during solid-phase peptide synthesis (SPPS) using standard coupling procedures. The probe is stable to TFA deprotection/cleavage mixtures containing ethanedithiol commonly used in Fmoc-SPPS. Furthermore, tritium may easily be introduced into the thiadiazole ring by base-catalyzed hydrogen-exchange. Upon irradiation at 245-300 nm, parent 1,2,3-thiadiazole rapidly eliminates N2, generating very reactive thioketene which reacts with amines to give a thioamide in high yield, even when the photolysis is carried out in hydroxylic solvents. In order to investigate the potential of the title compound as a heterobifunctional cross-linking reagent a model study with angiotensin II (AII) was conducted. The photoreactive peptide Nα-4-carbonyl-1,2,3-thiadiazole-AII (TDA-AII) was synthesized by Fmoc-SPPS and conjugated to bovine serum albumin (BSA) by photolysis at 245 and 300 nm. By use of a capture competition ELISA, the C-terminal Pro-Phe epitope of photoconjugated AII with the sequence DRVYIHPF was shown to bind specifically to antiAII antibodies (anti-AII abs), although antibodies against both the C- and N-terminal epitopes were present in the assay. A dipeptide His-Leu carboxy-extension form of AII, angiotensin I (AI), only bound to anti-AII abs at 100-200 times higher concentrations, showing that the C-terminal epitope was blocked by the dipeptide. © Munksgaard 1996.
 
Article
A new method of synthesizing ortho-methylated phenylalanines has been developed. Phenylalanines with at least one free ortho-position undergo a Pictet-Spengler cyclization with formaldehyde followed by hydrogenolytic splitting of the endocyclic benzylic C--N bond of 1,2,3,4-tetrahydroisoquinolines and afford corresponding ortho-methyl derivatives. Repeating this reaction sequence on the ortho-substituted phenylalanines yielded ortho,ortho-disubstituted derivatives, and para-substituted phenylalanines yielded ortho,para-disubstituted analogs. Our modified method of cyclization preserved the configuration at the chiral center: hydrogenolysis, on the other hand, led to racemization. Incorporation of the methylated phenylalanines into position 2 of oxytocin led to, in the case of the D-isomers, potent uterotonic inhibitors.
 
Article
Three Tic-containing (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) model peptides were synthesized to assess the tendency of this constrained Phe analogue to fold into a beta-bend and a helical structure, and to adopt a preferred side-chain disposition. The results of the solution conformational analysis, performed by using Fourier transform infrared absorption and 1H nuclear magnetic resonance, indicate that in chloroform the -Aib-D-Tic-Aib-, -(Aib)2-D-Tic-(Aib)2-, and -L-Pro-D-Tic- sequences fold into intramolecularly H-bonded forms to a great extent. An X-ray diffraction analysis on p-BrBz-(Aib)2-DL-Tic-(Aib)2-OMe monohydrate and p-BrBz-L-Pro-D-Tic-NHMe allows us to conclude that, while the pentapeptide methylester forms an incipient (distorted) 3(10)-helix, the dipeptide methylamide adopts a type-II beta-bend conformation. In both cases, the D-Tic side-chain conformation is D, gauche(-). The implications for the use of the Tic residue in designing conformationally restricted analogues of bioactive peptides are briefly discussed.
 
Article
An efficient method for solid phase peptide synthesis was developed, which consists of N alpha-selective deprotection by dilute methanesulfonic acid, in situ neutralization and rapid coupling reaction using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or 2-(benzotriazol-1-yl)oxy-1,3- dimethylimidazolidinium hexafluorophosphate (BOI) reagent. Selective removal of the N alpha-Boc group by dilute methanesulfonic acid was of more advantage than removal by TFA in terms of stability of semipermanent protecting groups and suppression of undesired side reactions. The use of in situ neutralization and rapid coupling method reduced intramolecular aminolytic cyclization by shortening exposure of the deprotected nucleophilic amino group. A successful synthesis of porcine brain natriuretic peptide (pBNP) has been achieved using this efficient solid phase peptide synthesis scheme.
 
Article
Cysteine is smoothly converted to S-3-sulphopropylcysteine by 3-hydroxypropane-sulphonic acid γ-sultone (1,3-propane sultone) in aqueous propanol at pH 8.3. The reaction is about 12 times slower than with iodoacetate but the selectivity for thiol groups is similar, as judged from model experiments and reactions with proteins. S-3-Sulphopropylcysteine is stable to the conditions to total acid hydrolysis and behaves distinctively in electrophoresis and ion-exchange chromatography. Reduction and alkylation of bovine insulin and human serum albumin (pH 8.3, 50% aqueous propanol) on a preparative or analytical scale gives fully S-substituted derivatives. The S-sulphopropylated A and B chains of insulin have been separated by precipitation or ion-exchange chromatography. The sulphopropyl derivatives have good handling properties and the reaction with propane sultone is suggested as a useful addition to the methods of protein chemistry.
 
Article
Derivatives of bovine growth hormone, containing monoaminotyrosyl residues in positions 35, 42 and 174, were treated at pH 3.6 with a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Under these conditions aminotyrosyl groups reacted. On changing the pH to 9.3, the second fluorine atom of the reagent was substituted with the sterically adjacent side groups of lysine, since the excess of reagent had been previously removed. The modified protein underwent cyanogen bromide treatment. Peptides containing the crosslinks were purified from tryptic digests of the cyanogen bromide fragments by HPLC. Results show that aminoTyr 174 was able to form dinitrophenylene bridges with Lys 111, Lys 29 and Lys 170. AminoTry 35 was found crosslinked to Lys 29. Taking into account the size of the reagent, it may be inferred that Lys 29, 111 and 170 are located at approximately 5 A from Tyr 174 in the bovine growth hormone molecule.
 
Article
RGD peptides are known as important ligands for integrin receptors in the cell adhesion process. The selectivity of RGD peptides for a certain integrin receptor is partly dependent on the RGD conformation and the residues surrounding the RGD sequence. This paper investigates the effect of the addition of a phenylalanine residue on the RGD conformation in cyclo(1,6)Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2 (1) as compared to the previously studied cyclo(1,5)Ac-Pen-Arg-Gly-Asp-Cys-NH2 (2). The conformational study of peptide 1 was done in aqueous solution using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations. This work will increase the understanding of the flanking residue's effect in RGD peptides.
 
Article
Tyrosine contributions to the structure-function relationship in the fructose-1, 6-biphospate aldolase from C. capitata have been investigated. There are three well defined groups of tyrosine residues with different roles in the structure of the insect aldolase. C-terminal tyrosine residues are essential for the maintenance of the catalytic conformation. Releasing of these residues by carboxypeptidase A treatment results in complex conformational changes according to CD studies. Another tyrosine residue group is located at the active site, and the substrate, fructose-1, 6-biphosphate, protects it upon nitration. Chemical modification of this residue results in enzyme activity changes similar to those induced by carboxypeptidase digestion. Enzyme-substrate interaction results in a change of the microenvironment of at least three tyrosine residues per subunit with different accessibility for tetranitromethane.
 
Article
The eicosapeptide (Gly88,90) 82-101 hCG-beta was synthesized by the fragment condensation of the nonapeptide (Gly88,90) 82-90 and the undecapeptide 91-101 followed by iodine oxidation to make the disulfide loop. Intramolecularity of the disulfide linking cysteines at 93 and 100 was confirmed. Antipeptide antibodies were elicited in rabbits upon immunization with a conjugate of the peptide with tetanus toxoid. The repertoire of these antibodies was directed solely against the undecapeptide 91-101. These antibodies showed no recognition for hCG, hCG-beta or ARCM hCG-beta, suggesting that the conformational epitopes of hCG-beta in the region 82-101 may not be accessible to antibodies.
 
Article
The peptide corresponding to the (66-104) sequence of horse heart cytochrome c and its carboxyamide analog, selectively modified at the critical Met80 residue, have been synthesized by stepwise solid-phase methods on PAM and BHA resins respectively. The correctness of the growing peptide chain as well as the homogeneity of the final products have been monitored by several analytical methods including quantitative Edman degradation. After HF cleavage both peptides were purified by semipreparative HPLC. The overall yields were 24% for the native (66-104) and 10% for the carboxyamide analog. The homogeneity of the purified synthetic peptides have been determined by different criteria including HPLC, amino acid composition, Edman degradation, electrophoresis, and tryptic peptide mapping. The synthetic fragments have been utilized for preliminary semisynthesis experiments with the native [Hse greater than 65] (1-65)H heme-sequence.
 
Article
The syntheses of some derivatives of horse cytochrome c-(66-79)-tetradecapeptide are presented. The syntheses are so designed that analogues of this phylogenetically well preserved sequence can be obtained also. The compounds were intended as synthons for the semisynthesis of 65-homoserine-cytochrome c, which we described earlier. A requisite for this project was the C-terminal tetracosapeptide fragment of the protein, accessible through degradation of cytochrome c with cyanogen bromide. The five epsilon amino groups in this compound are reversibly protected with the 2-(methylsulfonyl)ethyloxycarbonyl function, which is resistant to acid and causes little impairment of solubility. The condensation of the fragments leading to the native sequence of horse cytochrome c-(66-104)-nonatriacontapeptide is presented also. The syntheses were performed using the solution strategy. Some unexpected ring closing reactions involving tyrosine and tert.-butyl prolylasparaginylcarbazate, are described.
 
Article
A solution synthesis is described of the partially protected N alpha-benzyloxycarbonylheptadecapeptide Z-Lys (Tfa)-Thr-Glu-Arg-Glu-Asp-Leu-Ile-Ala-Tyr-Leu-Lys (Tfa)-Lys (Tfa)-Ala-Thr-Asn-Glu (OBu t)-OBu t corresponding to sequence 88-104 of horse heart cytochrome c. The synthesis is achieved through the preparation of two subunits H1 (sequence 88-96) and H2 (sequence 97-104) and their linkage by an azide coupling step.
 
Article
The synthesis by fragment condensation of a nonatricontapeptide corresponding to positions 66-104 of the proposed amino acid sequence of the enzyme ribonuclease T1 is described. It is shown that complex peptides corresponding to the C terminal region of the T1 enzyme are soluble in 8 M pH 10.7 urea and that purification can be achieved by gel filtration in this solvent.
 
Article
We have shown that two CNBr fragments of horse apocytochrome c, [Homoser-lactone65](1-65) and (66-104), bind to the ferric heme fragment (1-25)H to form a non-productive three-fragment complex, and that when the heme of this complex has been kept reduced for 48 h at 25 degrees, the peptide bond between residues 65 and 66 is restored with a yield of 24% or more. We have also shown that another CNBr fragment [Homoser-lactone65](23-65), but not [Homoser-lactone65](39-65), similarly rejoins to fragment (66-104) in the presence of the ferrous heme fragment with 25% or more yield. For complex of ferro-heme fragment [Hse-lacton65](1-65)H and apofragment (66-104) of horse cytochrome c, which restores the peptide bond between residues 65 and 66 (located on the left side of the heme) (cf. Harbury, H.A. (1978) in Semisynthetic Peptides and Proteins (Offord, R.E. & DiBello, C., eds.), pp. 73-89, Academic Press, New York). Corradin & Harbury have suggested that axial ligation of methionine 80 to the heme (on the left side) is important. Consistent with their idea, fragment [Hse80](66-104) was found not to link to [Hse-lactone65](1-65) in the presence of ferro(1-25)H. Furthermore, the present studies indicate that the interaction involving residues 26 to 38 (on the right side) is also important for such a conformation which assists in the rejoining of the two apofragments. Combining these two ideas, we suggest that restoration of the peptide bond between residues 65 and 66 reflects the structural integrity of these complexes in the reduced form. Thus, the present reaction system can be used not only for chemical synthesis of [Homoser65] apocytochrome c but also to extend amino acid substitution studies of cytochrome c to residues 1 to 64.
 
Article
DiMe-C7 (pGlu-Gln-Phe-MePhe-MeGly-Leu-MetNH2), a metabolically stable analogue of Substance P, was prepared by solid-phase peptide synthesis using a polyacrylamide resin and a labile anchorage derived from glycolic acid. Myotropic activities in guinea pig ileum (ED50 = 4.0 +/- 1.5 10(-8) M) and guinea pig trachea (ED50 = 8.6 +/- 3.5 10(-8) M) are discussed in comparison with the corresponding activities of Substance P.
 
Article
The antimicrobial peptide, lactoferricin, is generated upon the gastric pepsin cleavage of lactoferrin and has many basic and hydrophobic amino acid residues essential for its biological activity. To investigate the structure-antimicrobial activity relationships, the basic amino acid-rich region of bovine lactoferricin (BLFC), RRWQWRMKKLG, was selected. Using chemically synthesized BLFC and its substituted peptides, the antimicrobial activities of the peptides were tested by determining the minimal inhibitory concentration (MIC) of Escherichia coli and Bacillus subtilis and the disruption of the outer cell membrane of E. coli, and the peptide's toxicities were assayed by hemolysis. The short peptide (B3) composed of only 11 residues had similar antimicrobial activities while losing most of the hemolytic activities as compared with the 25 residue-long ones (B1 and B2). The short peptides (B3, B5 and B7) with double arginines at the N-termini had more potent antimicrobial activity than those (B4 and B6) with lysine. However, no antimicrobial and hemolytic activities were found in B8, in which all basic amino acids were substituted with glutamic acid, and in B9, in which all hydrophobic amino acids were substituted with alanine. The circular dichroism (CD) spectra of the short peptides in 30 mM SDS were correlated with their antimicrobial activities. These results suggested that the 11-residue peptide of BLFC is involved in the interaction with bacterial phospholipid membranes and plays an important role in antimicrobial activity with little or no hemolytic activity.
 
Article
The secondary structure of 11 mammalian growth hormones has been predicted by combining five different methods. Three long helical regions located around residues 20, 120, and 170 constitute the most prominent common feature in the species studied. The strong amphiphilic character of these helices suggests that they can play an important role in protein folding or stability.
 
Article
Analogues of [Orn6]-SP6-11 have been synthesized in which the CH2SCH3 group of Met11 is replaced by a COOCH3 or a COOBzl group. These analogues, which were tested for agonist and antagonist activity in three in vitro preparations representative of NK-1, NK-2 and NK-3 receptor types, were full agonists at NK-1 receptors, showed very weak agonist activity at NK-2, receptors and were weak antagonists at NK-3 receptors. The above analogues were modified by substituting the alpha-carboxamide of residue 11 by a COOCH3 and a COOBzl group, respectively. The resulting analogues were found to be devoid of agonist activity in each of the functional assays. However, they showed weak antagonist activity at each receptor subtype, with the exception of the dibenzyl analogue, which was a potent and selective NK-1 receptor antagonist. It is concluded that appropriate modification of the side chain of Met11 and its alpha-carboxamide leads to a potent and selective at NK-1 receptor antagonist.
 
Article
Two hexapeptide analogues of Substance P (6-11) have been synthesized. Replacement of Gly9 by proline provides a peptide with tenfold enhanced selectivity for the NK-1 receptor. The corresponding proline-containing glycopeptide incorporating a beta-D-glucopyranosyl residue linked to the side-chain of Glu6 was 100 times more selective than Substance P for the same receptor.
 
Article
Three analogs of human beta-endorphin (beta h-ER) were synthesized by the solid-phase method: [Gln8,Trp27]-beta h-EP (I), [Gln8,Arg9,Trp27]-beta h-EP (II), and [Gln9,Arg11,Trp27]-beta h-EP (III). Radioreceptor binding assay with use of tritiated beta h-EP as primary ligand gave relative potencies as follows: beta h-EP, 100;I, 778;II, 467;III, 449. Relative potencies in an analgesic assay were: beta h-EP, 100;I, 114;II, 165;III, 83. The 8-11 segment of beta h-EP can tolerate a net increase in charge of +2 without diminishing analgesic potency. The substitution of Glu8 may be one of the more dependable means of designing beta-endorphin antagonists.
 
Article
Synthesis is described of the protected tetrapeptide corresponding to positions 11-14 of the primary structure of the porcine pancreatic secretory trypsin inhibitor II (Kazal), in the form of free acid as well as protected hydrazide. The tetrapeptide tert-butyloxycarbonylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyl-lysine was prepared by stepwise elongation from the C-terminal Nepsilon-trifluoroacetyllysine using successively 1-succinimidyl benzyloxycarbonylprolinate, p-nitrophenyl N-tert-butyloxycarbonyl-S-acetamidomethylcysteinate and 1-phenyl-3-methyl-4-(tert-butyloxycarbonylglycyl)-oximinyl-5-(benzyloxycarbonylglycyl)-imino-2-pyrazoline as acylating agents. Alternately, the dipeptide benzyloxycarbonylprolyl-Nepsilon-trifluoroacetyllsine was transformed into the corresponding tert-butyloxycarbonylhydrazide which was reacted, after catalytic hydrogenolysis, with tritylglycyl-S-acetamido-methylcysteine to give the tetrapeptide tritylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyllsine tert-butyloxycarbonylhydrazide. The stereochemical homogeneity of the final products was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M, followed by quantitative amino acid analysis.
 
Article
Bovine growth hormone was modified by reaction with 1,5-difluoro-2,4-dinitrobenzene under conditions favouring production of intramolecularly crosslinked derivatives from monomeric molecules. The monomeric fraction, isolated by chromatography on Sephadex G-100, was oxidized or reduced and carbamidomethylated and trypsin digested. The resulting peptides were fractionated on SP-Sephadex and further purified by peptide mapping or HPLC. Two modified peptides containing sequences 108-112 or 108-113 and 171-176 of bGH were obtained, including a dinitrophenylene bridge between lysine 111 and tyrosine 174, thus suggesting the stereochemical proximity of these residues.
 
Article
The dodecapeptide sequence, Tyr-Gly-Gly-Phe-Met-Lys-Arg-Tyr-Gly-Gly-Phe-Met (BI), which is totally conserved in the primary structures of human, bovine, rat and toad preproenkephalins, has been synthesized by the solid-phase method. Coupling reactions were achieved by using symmetrical anhydrides of tert.-butyloxycarbonylamino acids performed with N-tert.-butyl,N'-methylcarbodiimide. 6-Arg and 7-Lys analogs have also been obtained. The peptides show opiate activity in both GPI and MVD assay, and possess antinociceptive properties as estimated by the hot-plate test in mice when applied intracisternally.
 
Article
RNase-(1-118) containing native disulfide bonds is similar in fold to native RNase A but not of lowest Gibbs energy as compared with the isomers containing non-native disulfide bonds. The present n.m.r. studies have indicated a dramatic increase in the exchange rate of all of the 'protected' amide protons of RNase-(1-118) over RNase A. A calculation shows a large increase in the rate of 'opening' of the structure. The exchange rate of the protected amide protons of RNase-(1-120) is slower than RNase-(1-118) but much faster than RNase A. Binding with a synthetic complementing fragment (114-124) markedly reduces the exchange rate of 20 to 25 amide protons of RNase-(1-118). It has previously been shown that binding with a complementing fragment of RNase-(1-118) generates a lowest Gibbs energy state. Thus, using available thermodynamic information for interpretation, we suggest that a) removal of six carboxy terminal residues of RNase A would disrupt coupling between these residues and those distant in the structure (loss of extra stabilizing energy), b) this would, in turn, alter the enthalpy-entropy compensation in such a way that the magnitude of Gibbs energy change favoring folding is significantly reduced without a large change of fold and c) in this activated state the molecule would be highly motile.
 
Article
The hexapeptide dimer (H-Hcy-Glu-His-Phe-D-Lys-Phe-OH)2 was synthesized using solution methods and characterized. Its conversion into H-Met(O2)-Glu-His-Phe-D-Lys-Phe-OH, Org 2766, was studied on a small scale in as short a time as possible; reduction of the disulfide bond using Na/NH3, reaction with CH3I, oxidation with H2O2 and catalyst and purification by HPLC were carried out starting with 2 mg of the dimer in a total preparation time of approximately 22 min, starting with the addition of CH3I. The preparation of the 11C-labelled analogue was carried out by methylation with 11CH3I. Restrictions imposed by working with carbon-11 will be discussed.
 
Article
11S globulin, the major storage protein of soybean seeds, was isolated to be homogeneous in ultracentrifugation, disc electrophoresis and isoelectric focusing by a comparatively simple method using gel filtration on Sephadex G-100 and G-200 columns with a good yield. The protein had the molecular weights of 309,000–322,000 by three separate methods based on different principles. Subsequently, some physico-chemical properties of the protein were determined. For example, sedimentation coefficient, isoelectric point, intrinsic viscosity and absorptivity constant were 12.2S, pH 4.64, 0.0485 dl/g and 8.04, respectively. Optical rotatory dispersion studies of the protein gave the values of a0 = —246 and b0 = —33. The contents of α-helix and β structure calculated from these parameters were estimated to be 5.2% and 34.8%, respectively.
 
Article
The molecular shape of the 11S globulin, the major storage protein of soybean seeds, was estimated to be an oblate ellipsoid with a revolution axial ratio of 8.11-8.38 with or without scarce hydration according to the procedure of Simha & Perrin by measuring the partial specific volume, diffusion coefficient, molecular weight and volume fraction intrinsic viscosity. The length of the major axis of the molecule is 178-180 A and that of the minor axis 22 A. Subsequently, the shape factor beta, and the hydrodynamically effective volume, Ve, of the protein were calculated by the procedure of Scheraga & Mandelkern. Consequently, the 11S globulin molecule was also an oblate ellipsoid from beta. Ve was found to be equal to MV/N, the anhydrous volume of protein. It was therefore concluded that the protein existed in a rigid and nearly anhydrous state in solution.
 
Article
[Tyr120]-RNase 111–124 and [Ala120]-RNase 111–124 were synthesized and purified to chromatographic and electrophoretic homogeneity. The peptides were mixed noncovalently with RNase 1–118 that had been prepared by enzymatic degradation of native bovine pancreatic ribonuclease A. The activities of the resulting complexes were compared with that of the corresponding complex containing the natural peptide sequence, [Phe120]-RNase 111–124, and with native RNase, using yeast RNA, C > p and U > p as substrates. The Tyr120 and Phe120 complexes were equally active against RNA and C > p, but the Tyr120 complex was twice as active as the Phe120 complex against U > p. The complex with alanine at position 120 was only 1% as active toward C > p as the complex containing phenylalanine. The prediction that giraffe RNase, which contains tyrosine at position 120, would be relatively more active toward U > p than C > p compared with bovine RNase was verified.
 
Article
The molecule of thermolysin was shown by X-ray crystallography to be composed of two structural domains of equal size comprising residues 1-157 and 158-316. In order to explore the possibility that these domains correspond to globular fragments able to refold autonomously, we have investigated the conformational and stability properties of fragment 121-316, which was obtained by limited chemical cleavage of thermolysin with cyanogen bromide. As judged by far-ultraviolet circular dichroism measurements, in aqueous solution under neutral conditions the fragment maintains a relative amount of helical structure which is comparable to that exhibited by the corresponding region in native thermolysin. The secondary structure attained by the fragment appears remarkably stable to the denaturing action of heat. By measuring the temperature dependence of the dichroic signal at 220 nm a Tm near 74 degrees was obtained. Immunodiffusion analyses indicated that the fragment recognizes and precipitates antibodies raised in rabbits using native thermolysin as immunogen. The overall conformational and immunochemical data indicate that fragment 121-316 of thermolysin is able to refold into a stable structure of native-like characteristics independently of the rest of the molecule. The results of this study complement those previously reported for fragment 206-316 (Vita, C., Fontana, A., Seeman, J.R. & Chaiken, I.M. (1979) Biochemistry 18, 3023-3031).
 
Article
A nonapeptide, Arg-Glu-Leu-Glu-Asp-Gly-Thr-Pro-Arg, corresponding to the 123–131 sequence of bovine pituitary growth hormone, was prepared by a modified solid phase synthesis. To avoid an acid- or base-catalysed rearrangement at the Asp-Gly bond, which gives rise to an aspartimide-containing peptide, a new combination of protecting groups was introduced. The 2-(4-biphenylyl)-2-propyloxycarbonyl group was used for Nα-protection, a t-butyl ester for the β-carboxyl of aspartic acid, and a p-alkoxybenzyl alcohol ester for the anchoring bond to the resin support. The nonapeptide showed growth hormone activity in the hypophysectomized rat tibia epiphyseal width test, but was inactive in the hypophysectomized rat body weight gain test.
 
Article
The solubility prediction method for protected peptides was successfully applied to relatively small peptide fragments of human hemoglobin alpha-chain (123-136) which contained various polar amino acid residues such as Asp(OBzl), Glu(OBzl), Lys(Z), Ser(Bzl), and Thr(Bzl). As reported previously for hydrophobic peptides and human proinsulin C-peptide fragments, solubility data indicated that the insolubility of protected peptides having a mean value of Pc value below 0.90 appeared to begin at the octa- or nonapeptide sequence level and that beta-sheet structure played an important role in the insolubility of peptides. When a peptide has a beta-sheet structure in the solid state, we can clearly determine the critical chain length for peptide insolubility, the solubility dependence on solvent properties, and the solubility independence of amino acid compositions of peptides.
 
Article
In the course of our study concerning gastrin and cholecystokinin (CCK) receptors, we have synthesized and characterized a new labeled gastrin ligand, 125I-BH-[Leu15]-gastrin-(5-17) [(3-[125I]iodo-4-hydroxyphenyl)-propionyl-[Leu15]-gastrin-(5-17)]. Binding of 125I-BH-[Leu15]-gastrin-(5-17) to isolated canine fundic mucosal cells was specific, saturable and of high affinity. 125I-BH-[Leu15]-gastrin- (5-17) and 125I-BH-CCK-8[(3-[125I]iodo-4-hydroxyphenyl)-propionyl-CCK-8] interact with isolated canine fundic mucosal cells with small differences in maximal binding capacities and affinities, 3800 +/- 900 binding sites/cell (Kd = 0.52 +/- 0.23 nM) and 6200 +/- 1100 binding sites/cell (Kd = 0.31 +/- 0.18 nM), respectively. The relative order of potencies for gastrin and CCK analogs in displacing 125I-BH-[Leu15]-gastrin-(5-17) binding correlated well with those obtained using 125I-BH-CCK-8. Selective CCK/gastrin antagonists L-364,718 (MK-329) and L-365,260 also inhibited 125I-BH-[Leu15]-gastrin-(5-17) binding. These results indicate that 125I-BH-[Leu15]-gastrin-(5-17) binds to gastrin receptors in isolated canine fundic mucosal cells. We have also characterized 125I-BH-[Leu15]-gastrin-(5-17) binding to the human Jurkat lymphoblastic cell line (Jurkat cells) known to express the CCK-B/gastrin receptor. Saturation experiments have shown that both 125I-BH-[Leu15]-gastrin-(5-17) and 125I-BH-CCK-8 interact with a single class of high-affinity binding sites in the Jurkat cell line.(ABSTRACT TRUNCATED AT 250 WORDS)
 
Article
A cyclic hexapeptide cyclo(Lys-Gly-Asp-Gln-Leu-Ser-) 10 was synthesized stepwise in solution by acylation of peptide ester trifluoroacetates directly with preactivated Boc-amino acids using the DCC/HOBt method; the final cyclization reaction was performed using the pentafluorophenyl ester method in solution (1-4). This peptide is a cyclic derivative of murine tumor necrosis factor-(127-132) and is designed as a potential antitumor agent. The cyclic peptide 10 displayed weak cytotoxic activity on three of the four human tumor cell lines tested.
 
Article
Crystals of [Phe4 Val6] antamanide (cyclic [ValProProPhePhe]2) grown from dioxane/H2O, with space group P21212 and cell parameters a = 15.099(4), b = 22.008(5) and c = 11.024(3) A, are almost identical to crystals grown from H2O/acetone, the structure of which was determined a number of years ago. Per peptide molecule there are the equivalent of 12 water molecules occupying 16 sites in both crystals; however, in the new investigation a number of water molecules present at one-half occupancy have been found in different positions than in the earlier analysis. The interpretation of the hydrogen bonding between peptide/water and between water/water is much more satisfactory. Pentagonal water assemblies are present in the solvent channel. There is a distinct indication of the occurrence of a bifurcated bond between two water molecules, as well as the presence of three-center hydrogen bonds joining three water molecules. This may be the first experimental example of a bifurcated bond between two water molecules.
 
Article
The 12S globulin, one of the major storage proteins of rapeseeds, has the following physico-chemical constants, as determined by ultracentrifugation, quasi-elastic light scattering measurements and gel chromatography: sedimentation coefficient S20(0), w = 12.7 x 10(-13) s; diffusion coefficient (quasi-elastic light scattering) D20(0), w = 3.8 x 10(-7) cm2 S-1; Stokes radius (by quasi-elastic light scattering) Rs = 5.7 nm and (by gel chromatography) Rs = 5.5 nm; partial specific volume (calculated from the amino acid composition) v(-) = 0.729 ml g-1; molecular weight Ms, D = 300,000 daltons, Ms, Rs = 294,000 daltons (Rs from the gel chromatography); frictional ratio f/fo = 1.28.
 
Article
The effect of SDS on the 12S protein fraction of mustard seed (B. juncea) has been followed by the techniques of ultracentrifugation, gel filtration, gel electrophoresis, viscosity, ultraviolet difference spectra and fluorescence spectra. At low concentrations of SDS, up to 0.1%, both aggregation and dissociation of the protein occurs. Only dissociation occurs at higher SDS concentrations and is complete at 0.5% SDS. Viscosity increases sharply up to 0.15% SDS, remains constant between 0.15 and 0.30% and then increases markedly again. SDS induces also difference spectra with minima at 280, 288 and 295 nm. Fluorescence emission intensity increases at SDS concentrations less than 0.05% and quenching occurs at higher SDS concentrations. The results suggest that SDS causes association, dissociation and denaturation of the protein molecule.
 
Article
The effect of low pH on the molecular properties of mustard 12S protein has been studied by the techniques of ultracentrifugation, viscometry, electrophoresis, turbidimetry, u.v. difference spectroscopy, fluorescence spectroscopy and circular dichroism. Ultracentrifugation and electrophoresis experiments indicated dissociation of the protein in the pH range 5.0 to 3.0 and below this pH reaggregation was indicated. Viscosity, turbidimetry, u.v. difference spectroscopy, fluorescence spectroscopy and circular dichroism studies showed that denaturation of the protein occurred between pH 5.0 and 3.0 and refolding at pH values below 3.0.
 
Article
Dynorphin-(1-13) (Dyn-(1-13)) and various analogs substituted in positions 8 and 10 were synthesized by the solid-phase technique and analyzed for their ability to inhibit the electrically evoked contraction of the guinea pig ileum (GPI) and to compete with the binding of [3H]-ethylketocyclazocine (EKC, kappa ligand), [3H]-[D-Ala2, MePhe4-Gly-ol5]-enkephalin (DAGO, mu ligand) and [3H]-[D-Ser2, Thr6]-Leu-enkephalin (DSLET, delta ligand) to membrane preparations of the guinea pig cerebellum or rat brain. Introduction of Ala in position 8 decreased the activity of the peptide on the GPI by 50% but induced a 2.22-fold increase in its affinity for the kappa receptor ([3H]-EKC binding displacement from guinea pig cerebellum; Ki of 0.05 nM as compared with 0.11 nM for Dyn-(1-13)). On the other hand, the ability of [Ala8] Dyn-(1-13) to displace the binding of [3H]-DSLET from rat brain membranes was decreased by a factor of 1.7 while its affinity for the mu receptor was not greatly affected ([3H]-DAGO displacement; Ki of 0.44 nM as compared with 0.50 nM for Dyn-(1-13)). Replacement of position 8 by D-Ala caused similar changes in the activity of the peptide but the increase in its affinity for the kappa site was somewhat smaller (Ki of 0.08 nM as compared with 0.11 nM). [D-Pro10]-Dyn-(1-13) was equipotent to [Ala8]-Dyn-(1-13) in the GPI but its affinity for the mu binding site was decreased by a factor of 2.7 as compared with Dyn-(1-13).(ABSTRACT TRUNCATED AT 250 WORDS)
 
Article
The synthesis of three collagen model analogs is described: Ac-Ala-Gly-Pro-Ala-Gly-Pro-NHMe, Ac-Ala-Gly-Pro-Ala-Glc-Pro-NHMe, and Ac-Ala-Glc-Pro-Ala-Gly-Pro-NHMe, where Glc stands for glycolic acid. The 1H-n.m.r. properties of these compounds in dimethylsulfoxide-d6 and trifluoroethanol are described. While in DMSO-d6 the compounds are random, in TFE the glycine amide protons seem to be less solvent exposed than the other amide protons. Little difference was found in the behavior of the three compounds.
 
Article
The acid-base titration of bleomycin-A2 in D2O solution at 35 +/- 5 degrees has been monitored by 13C n.m.r. spectroscopy at 67.89 MHz. The following pKDa values were obtained: 3.68 +/- 0.05 (secondary amine), 5.29 +/- 0.03 (imidazole), and 8.23 +/- 0.19 (primary amine), where KDa is the dissociation constant in D2O solution. The equilibrium isotope effects (pKDa--pKa in H2O) are: 0.70 +/- 0.06 (secondary amine), 0.28 +/- 0.04 (imidazole), and 0.85 +/- 0.19 (primary amine). Titration of the imidazole group of Bleo-A2 occurs at Npi, i.e. only Ntau is protonated in basic solution. Significant protonation shifts are almost completely limited to carbons of the N-terminal tetrapeptide, suggesting that the C-terminal tripeptide extends into the solvent and interacts to a minimal extent with the rest of the molecule. Long range protonation shifts associated with titration of the imidazole and secondary amine groups indicate that protonation of one or both of these sites is probably accompanied by significant conformational changes. The observed protonation shifts generally fail to correlate with Zn(II) complexation shifts reported by Dabrowiak et al. (1973, Biochemistry 17., 4090) indicating that ligation sites cannot unambiguously be determined from these complexation shifts. The complexation shifts previously attributed to coordination of the imidazole and carbamoyl groups probably result from conformational changes.
 
Article
The 13C-D-Leu12, 14 gramicidin A was synthesized by the solid phase method incorporating 13C-D-leucine in positions 12 and 14 with about 25 and 50% enrichment, respectively. The pentadecapeptide was removed from the resin by ethanolamine treatment, with the N-protecting group (Boc) still on. After removal of the protecting group, the peptide was formylated and purified by preparative t.l.c. to obtain 13C-D-Leu12, 14 gramicidin A in a very pure state in an overall yield of about 12.5%. The peptide was then thoroughly characterized by HPLC which gave one single peak with the same retention time as that of Val1-gramicidin A of the natural gramicidin mixture. The CD spectra of the synthetic and the HPLC purified natural Val1-GA were obtained and found to be identical, indicating the optical purity of the sample. The synthetic GA was characterized by 13C n.m.r. spectrum and compared with that of natural GA. Single channel conductance parameters of the synthetic GA were determined and found to be indistinguishable from those of natural Val1-GA in lipid bilayer membranes and the mean channel lifetime was found to be as reported earlier by others.
 
Article
Conformational features of dynorphin A-(1-13) were examined by laser Raman spectroscopy. Dynorphin A-(1-13) appears to have a mixture of extended beta-pleated sheet and "random" structure.
 
Article
We have synthesized both a protected nonapeptide of the mycobacillin 8-13-1-3 amino acid sequence and a protected tridecapeptide of the 4-13-1-3 sequence, which are a fragment and a open chain analog of this antibiotic, respectively. Some of their analogs with a reversed configuration of the amino acids at fixed positions have also been synthesized. The nonapeptides were obtained by coupling partially protected mycobacillin fragments with the sequence 8-10 and 11-13-1-3 while the tridecapeptides were synthesized by coupling partially protected fragments 4-7 and 8-13-1-3. Configuration analogs of these fragments were also used. The coupling methods applied were DCCI/HONSu or DCCI/HOBt. The purification of the synthesized peptides was achieved by means of recrystallization or column chromatography on silica gel. They were characterized mainly by m.p., degree of optical rotation, elemental and amino acid analysis.
 
Article
Three 2-acetamido-2-deoxy-alpha-D-galactopyranoses attached to Ser2, Thr3 and Thr4 of the amino-terminal portion of glycophorin AM are responsible for the so-called TN blood group specificity. The corresponding glycopeptide H2N-Ser-Ser*-Thr*-Gly-OH obtained by a stepwise peptide coupling strategy was submitted to a detailed high-field nuclear magnetic resonance (n.m.r.) analysis. 13C-n.m.r. spectrum confirms the validity of previous assignments made on M sialo and asialoglycopeptides obtained by specific degradation of human glycophorin AM. In addition, the 400 MHz 1H-n.m.r. spectrum allowed most of the proton resonances to be assigned. A careful examination of the chemical shifts and coupling constants revealed some interesting features of the conformational properties of the GalNAc-Ser and GalNAc-Thr linkage as well as of the rotational isomerism of Thr and Ser side-chains. The data give conclusive evidence that high-field n.m.r. spectroscopy can be successfully used to gain structural and dynamic information on rather sophisticated glycopeptides.
 
Top-cited authors
Gregg B Fields
  • The Scripps Research Institute
Arpad Furka
  • Eötvös Loránd University
Gábor Dibó
  • Eötvös Loránd University
Nobuhiro Go
Gregg B Fields
  • Florida Atlantic University