Tryptophan Residues In Proteins Research Articles

The Effect of Selective Modification of Tryptophan, Lysine and Arginine Residues of Basic Brain Protein on Encephalitogenic Activity

Abstract Basic protein (BP), which caused experimental allergic encephalomyelitis in guinea pigs in a dose of 30 μg, was isolated from bovine brain. Studies were carried out to characterize the encephalitogenic determinant by means of selective and permanent modification of tryptophan, lysine and arginine residues of BP. When tryptophan residues of BP were altered by the addition of 0.045 μmol 2-hydroxy-5-nitrobenzyl bromide/mg BP, preparations exhibited a significant decrease in encephalitogenic activity. In contrast, acetylation of free amino groups of lysine with acetic anhydride, or modification of guanidinium groups of arginine with 2,3-butanedione, had no effect on the encephalitogenic activity of BP. These results suggest that tryptophan is an essential component of the encephalitogenic determinant, whereas lysine and arginine are probably not present in this site.

Experimental allergic encephalomyelitis: synthesis of disease-inducing site of the basic protein.

A highly encephalitogenic peptide whose structure resembles the sequence of amino acids surrounding the single tryptophan residue in the encephalitogenic A1 protein from bovine myelin was synthesized. This peptide is similar in the sequence to peptic peptide E and tryptic T27, derived directly from the A1 protein, and is as active on a molar basis as the A1 protein. The major disease-inducing site of the A1 protein resides in a linear sequence of nine amino acids: H-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH. This region of the A1 protein is apparently the major encephalitogenic determinant since specific modification of the tryptophan residue in the A1 protein with 2-hydroxy-5-nitrobenzyl bromide destroyed its encephalitogenic activity.

Coenzyme binding in yeast pyruvate decarboxylase. A fluorescent enzyme-inhibitor complex.

The formation of a fluorescent complex between apopyruvate decarboxylase of Saccharomyces carlsbergensis and thiochrome pyrophosphate, a competitive inhibitor with thiamine pyrophosphate, and Mg(II) ions is reported. A similar complex between thiochrome and the apoenzyme could not be detected, demonstrating the importance of the pyrophosphate group of the coenzyme in binding to the protein. Complex formation between the apoenzyme and thiochrome pyrophosphate in the absence of Mg(II) could also not be demonstrated. The role of magnesium in coenzyme binding is discussed, especially in reference to interaction with the pyrophosphate moiety. The apparent Km for Mg(II) ions, after correction for residual apoenzyme activity, is reported to be 87 μM.The shift in the fluorescence emission spectrum of thiochrome pyrophosphate of 23–24 nm toward lower wavelengths, upon complex formation, coincided with the behavior of thiochrome in solvents of lower polarity than water. A hydrophobic region at the coenzyme binding site of pyruvate decarboxylase is proposed. Enhancement of thiochrome fluorescence occurred concomitantly with the blue shift in the emission spectrum upon complex formation.The excitation wavelength of 290 nm employed to demonstrate complex formation suggested energy transfer from the tryptophan to the thiochrome chromophore. Quenching of fluorescence in the 340–350 nm region, however, occurred not only in the protein emission spectrum upon complex formation, but also when free tryptophan and thiochrome interacted with each other. The quenching of the protein fluorescence appears to be independent of hydrophobic interaction at the coenzyme binding site. It is suggested that protein tryptophan residues relatively exposed to solvent interact individually with the thiochrome molecule by an energy transfer mechanism to produce the quenching in pyruvate decarboxylase.

Sulfenyl Halides as Modifying Reagents for Polypeptides and Proteins

The determination of tryptophan and cysteine content in proteins by means of the selective reaction of sulfenyl halides with these amino acids has been carefully investigated. By reaction with 2‐ or 4‐nitrophenylsulfenyl chloride in aqueous acetic or formic acid, the nitrophenyl chromophore is covalently bound to the tryptophan and the cysteine residues of the protein, giving a thioether and a mixed disulfide respectively. The cysteine content has been determined spectrophotometrically at 450 nm from the 4‐nitrothiophenol released from the mixed disulfides of the cysteine residues after exposure of the 4‐nitrophenylsulfenyl chloride‐labelled protein in 0.1 N NaOH (at this wavelength the corresponding chromophore covalently bound to tryptophan does not absorb). Tryptophan has been estimated in the sulfenylated proteins by spectrophotometric determinations at 365 or 328 nm of the 2‐ and 4‐nitrophenylsulfenyl chromophores (ɛM 4000 and 11700 respectively), following removal of the yellow label from the cysteine residues, by dissolving the modified protein in 0.1 N NaOH. The procedure has been shown to be accurate for both amino acids with a wide variety of proteins.

Conformational studies on a modified poly‐L‐tryptophan: Circular dichroism and optical rotatory dispersion of poly‐2‐(2‐nitrophenylsulfenyl)‐L‐tryptophan and of random copolymers of L‐tryptophan and 2‐(2‐nitrophenylsulfenyl)‐L‐tryptophan

AbstractOptical rotatory dispersion (ORD) and circular dichroism (CD) measurements were carried out on a block copolymer, (γ‐ethyl DL‐glutamate)160 (L‐Trp)32, in which the tryptophan sequence has been modified to various extents by using 2‐nitrophenylsulfenyl chloride. The CD spectrum of the completely modified copolymer exhibits bands in some of the regions of maximum absorption of the sidechain chromophores. In the peptide absorption region the spectrum is similar to that reported in the literature for polypeptides in the α‐helical conformation. When the extent of modification of the tryptophan sequence is progressively reduced, there is a gradual change in the ORD spectra of the copolymers. On the basis of these data the assumption was made that no conformational change occurs on proceeding from the pure unmodified tryptophan sequence to the completely modified sequence. The results are discussed in connection with the study of possible conformational effects arising from selective chemical modification of tryptophan residues in proteins.

Allergic encephalomyelitis: Cleavage of the C-tryptophyl bond in the encephalitogenic basic protein from bovine myelin

The presence of one tryptophan residue in the encephalitogenic A1 protein from bovine spinal cord was established by six independent techniques. A nonencephalitogenic peptide containing 55 amino acid residues was derived from the A1 protein by cleavage of the C-tryptophyl bond with N-bromosuccinimide. This peptide, referred to as peptide T, was purified by gel filtration and Cellex-P chromatography and obtained in 31% yield. It appeared homogeneous by polyacrylamide gel and paper electrophoresis. The structure of peptide T was partially determined as follows: H-Gly-Ala-Glu-Gly-Gln-Ser-Pro-Phe-Gly-Lys….Ala-Arg-Arg-OH. The amino acid sequence of the N-terminal region is identical with that portion of the sequence of the encephalitogenic peptide E, a peptide obtained from pepsin hydrolysis of the A1 protein, which contains a tryptophan-glycine linkage. The H-Ala-Arg-Arg-OH sequence, established with carboxypeptidases A and B, is identical with that of the A1 protein and thus identifies peptide T as the 55 residues of the C-terminal region of the A1 polypeptide chain. The molecular weight of 5750 daltons was determined by amino acid analysis and confirmed by peptide mapping of the tryptic digest which showed 13 peptides (12 expected), 5 of which contain arginine. Peptide T, like the A1 protein, is highly basic and contains a total of 13 lysine, histidine, and arginine residues. The molecular weight falls close to that determined by quantitation of the PTH-glycine obtained by Edman degradation. Peptide T does not induce experimental allergic encephalomyelitis, even at 100 μg per guinea pig, and further emphasizes the importance of the tryptophan residue and the adjacent polypeptide region for determining encephalitogenic activity.

Differentiation between tryptophan residues in proteins.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDifferentiation between tryptophan residues in proteinsYehudit ElkanaCite this: J. Phys. Chem. 1968, 72, 10, 3654–3661Publication Date (Print):October 1, 1968Publication History Published online1 May 2002Published inissue 1 October 1968https://doi.org/10.1021/j100856a050RIGHTS & PERMISSIONSArticle Views52Altmetric-Citations27LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts

On the tryptophan determination in lysozyme and its photoxidation products

A comparative study was made of three techniques of tryptophan determination in lysozyme and its photoxidation products: mild acid hydrolysis (85–90°, 15 hr), pronase hydrolysis, and direct spectrophotometric determination. Satisfactory data have been obtained only after pronase hydrolysis. The technique for hydrolyzate treatment and its analysis have been worked out. This technique was used for tryptophan determination in three lysozyme photoxidation products. The mild acid hydrolysis has been found unsuitable owing to incomplete hydrolysis. The direct spectrophotometric determination has been modified as applied to lysozyme, and satisfactory results have been obtained. But this technique is unsuitable for tryptophan determination in photoxidized lysozymes as it does not permit differentiation of native from photoxidized tryptophan residues in protein.

A Colorimetric Procedure for the Quantitative Determination of Tryptophan Residues in Proteins

A simple method for the determination of tryptophan residues in proteins is described. The method involves (a) treatment of the protein with an excess of 2-hydroxy-5-nitrobenzyl bromide, (b) purification of the labeled protein by gel filtration and ethanol washing, (c) measurement of the absorbance at 410 mµ to determine the number of colored groups incorporated, and (d) a determination of the total protein content by conventional methods. The procedure has been shown to be accurate with a wide variety of proteins.

Studies on the action of diethyl pyrocarbonate on proteins

During the last few years the use of diethyl pyrocarbonate for cold-sterilization has drawn a lot of interest. Very little, however, has been done to elucidate the mechanism by which it acts on biological materials. In this paper the results of some enzyme experiments are reported, as well as studies on the action of diethyl pyrocarbonate on a model protein, bovine serum albumin. From data on fluorescence and absorbance changes the conclusion is drawn that diethyl pyrocarbonate reacts with tryptophan residues in proteins, and, from changes noted in the isoelectric point and the titration curve, that free amino groups are attacked. An essential reason for the inactivation of enzymes caused by diethyl pyrocarbonate, however, seems to be the production of irreversible, structural modifications manifested as a precipitation of the treated protein at pH values at or below the isoelectric point.

States of amino acid residues in proteins: V. Different reactivities with H 2O 2 of tryptophan residues in lysozyme, proteinases and zymogens

Hydrogen and organic peroxides were examined in a search for a reagent to differentiate between various states of tryptophan residues in proteins. Hydrogen peroxide in a solution of 0.5 M bicarbonate buffer (pH 8.1–9.4) containing 10% dioxane was found to possess a moderate oxidizing power to oxidize free and bound tryptophan residues at different concentrations of H 2O 2, and the strength of the tryptophan absorption band of the protein was lowered stepwise with increasing concentration. By the use of this reagent, tryptophan residues in several proteins were classified into various types with different oxidizabilities, and the moles, n, of each type per mole of proteine were determined; n=5 and 1 for lysozyme (EC 3.2.1.17), n=5, 1 and 1 for chymotrypsinogen and α-chymotrypsin (EC 3.4.4.5), and n=2, 1 and 1 for trypsinogen and trypsin (EC 3.4.4.4.) which are arranged in the order of decreasing oxidizability. On the activation of chymotrypsinogen, the oxidizability of the most strongly bound residue decreased greatly whereas, on the activation of trypsinogen, the oxidizability of the secondly oxidized residue increased slightly. The structural rearrangement on the activation was discussed in terms of these changes in state of tryptophan as well as those of other amino acids determined previously. The enzymic activity of lysozyme an proteinases was also measured as a function of H 2O 2 concentration, and the results were correlated with the degree of oxidation of tryptophan residues. All of the tryptophan residues in denatured proteins were oxidized at a low concentration of H 2O 2, so that the reagent is applicable for the determination of the molar tryptophan content in protein.