Modification Of Tryptophan Residues Research Articles

Modification of Tryptophan Residues on Rhizopus delemar C-Lipase Binding Chlorinated Pesticide by 2-Hydroxy-5-nitrobenzylation.

Comparative studies were carried out on the interaction of Rhizopus delemar C-lipase with 1, 1, 1-trichloro-2, 2-bis(4-chlorophenyl)ethane (DDT), 2, 2-bis(4-chlorophenyl)ethane (DIM), dichlorobenzophenone (DCBP) and aldrin by means of 2-hydroxy-5-nitrobenzylation of tryptophan (Trp) residues on the 1 : 1, 2 : 1 and 9 : 1 (7 : 1 with aldrin) pesticide-lipase complexes.2-Hydroxy-5-nitrobenzylation was carried out under three conditions : modification with a water-soluble modification reagent, with the same reagent in the presence of olive oil emulsion and with a fat-soluble modification reagent in the presence of emulsion. The Trp residues involved in ligand-binding were specified in tems of their modification patterns.Modification revealed that the binding of pesticide involves five exposed Trp residues. The modification patterns are distinctly different depending on the sort of pesticide. This is consistent with the previous observation that the binding of the above four pesticides affects the binding properties of the lipase quite differently depending on the sort of pesticide. It is suggested for DDT, DIM and DCBP that the binding of the first pesticide molecule, which governs the ensuing complex formation, involves the same Trp residue. This would indicate the presence of three overlapping binding sites for each of three pesticides. On the other hand, firstly binding two aldrin molecules bind to a region not involving a Trp residue.

Influence of Tryptophan Modification upon Digestion of Antithrombin III by Elastase

Chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNBSB) generates products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl [HNB] incorporated/mole of antithrombin III) but with high or low affinity for heparin-Sepharose. Upon digestion with pancreatic or neutrophil elastase the low affinity forms generate a product of molecular weight form (55 kDa) not seen in digests of native antithrombin III or modified forms with high affinity for heparin. When measured as loss of activity the observed rate of digestion of the latter in the absence of heparin was more rapid than that of native antithrombin III. The differences in digestion are considered to be related to conformation at differences between the various forms.

Chemical modification studies on a blood group A-specific lectin, crotalarin (Crotalaria striata) and its effect on hemagglutinating activity

Crotalarin, the N-acetyl-D-galactosamine-binding blood group A-specific lectin from the seeds of Crotalaria striata was subjected to various chemical modifications in order to ascertain the amino acid residues responsible for its carbohydrate-binding property. Modification of lysine, cysteine and arginine residues did not affect the carbohydrate-binding activity of the lectin. However, modification of tyrosine residue and carboxy group of the acidic amino acids led to a complete loss of its activity, indicating the involvement of tyrosine and aspartic and glutamic acid in the saccharide-binding respectively. The hemagglutinating activity of the lectin was completely/almost completely lost by modification of tryptophan residues. The relative loss in hemagglutinating activity on modification of tryptophan residues indicate that one residue/molecule is required for the carbohydrate-binding activity of the lectin. Modification was not effective in the presence of D-galactose (0.2 M). A marked decrease in the fluorescence emission was found as the tryptophan residues of crotalarin were modified. The c.d. spectra showed the presence of an identical pattern of conformation in the native and modified lectins which confirms that the loss in activity was due to modification only. The effect of periodate oxidation on crotalarin showed loss of activity whereas action of enzymes retained most of the activity.

Identification of the tryptophan residue present in the low affinity saccharide-binding site of ricin E.

The tryptophan residue located at the low affinity saccharide-binding site (LA-site) of ricin E was identified using two derivatives of ricin E, in which tryptophan residues were oxidized with N-bromosuccinimide (NBS) in the absence and presence of lactose. From the lysyl endopeptidase and tryptic digests of the B-chains of the respective derivatives, peptides containing oxidized tryptophan residue(s) were isolated by gel filtration on Bio Gel P-2 and HPLC. Analysis of the peptides containing oxidized tryptophan showed that tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative, in which the saccharide-binding ability at the LA-site was abrogated by modification of tryptophan residues with NBS in the absence of lactose. On the other hand, two tryptophan residues at positions 93 and 160 in the B-chain were found to be oxidized in the active derivative that retained the saccharide-binding ability at the LA-site after NBS-oxidation in the presence of lactose,...

2-Hydroxy-5-nitrobenzyl bromide as a specific reagent for tryptophan residues in membrane proteins: bacteriorhodopsin as an example.

The use of 2-hydroxy-5-nitrobenzyl bromide for the modification of tryptophan residues in integral membrane proteins is exemplified by its application to bacteriorhodopsin from Halobacterium halobium. Complete elimination of the unreacted reagent requires delipidation of the sample with detergents and posterior chromatography. This method also allows separation of the modified from the unmodified bacteriorhodopsin molecules. Modified molecules have lost the retinal, and are thus bleached, whereas the unmodified molecules appear to retain all the characteristics of solubilized native bacteriorhodopsin.

Modification of tryptophan residues during acidolysis of 4-methoxy-2,3,6-trimethylbenzenesulfonyl groups. Effects of scavengers

The formation of by-product 1 of tryptophan during the cleavage of Mtr from arginine can be minimized by addition of 1,2-ethanedithiol. Under too drastic conditions, however, a dithioketal product 2 is formed.

A tryptophan residue is essential to the sugar-binding site of winged bean basic lectin

Winged bean basic lectin contains four tryptophan residues per molecule (Molecular weight 53 000). Treatment of the lectin with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide cause a concomitant loss of the hemagglutinating activity with modification of tryptophan residues. Modification of two tryptophan residues out of original four caused a complete loss of the activity. The remaining two tryptophan residues remained unoxidized under non-denaturing conditions. Although N-bromosuccinimide also oxidized tyrosine residues, the nonparticipation of this amino acid in the activity was confirmed by acetylation of the lectin with N-acetylimidazole. The change correlated to oxidation of tryptophan residues by N-bromosuccinimide was prevented by d-galactose, the sugar specific for the hemagglutination of the lectin. These results clearly suggested that the loss of the hemugglutinating activity was due specifically to oxidation of tryptophan residues. The protected tryptophan residue was calculated to be one per molecule, indicating that a single tryptophan residue was essential to the sugar-binding site of the lectin. Moreover, modification of tryptophan residue was shown by an immunological study to produce no gross change in the conformation of the protein.

Chemical modification of tryptophan residues in castor bean hemagglutinin.

Modification of tryptophan residues in castor bean hemagglutinin (CBH) with N-bromosuccinimide (NBS) was investigated in detail. Tryptophan residues accessible to NBS increased with lowering pH and six tryptophan residues/mol were oxidized at pH 3.0, while two tryptophan residues/mol were oxidized at pH 5.0. From the pH-dependence curve for tryptophan oxidation, we suggest that -the extent of modification of tryptophan in CBH is influenced by an ionizable group with pKa=3.6. The saccharide-binding activity was decreased greatly by modification of tryptophan concomitantly with a loss of fluorescence. A loss of the saccharide-binding activity was found to be principally due to the modification of two tryptophan residues/mol located on the surface of the protein molecule. In the presence of raffinose, two tryptophan residues/mol remained unmodified with retention of fairly high saccharide-binding activity. The results suggest that one tryptophan residue is involved in each saccharide-binding site on each B-chain of CBH.

Chemical modification of dipeptidyl peptidase IV: Involvement of an essential tryptophan residue at the substrate binding site

Inactivation of pig kidney dipeptidyl peptidase IV (EC 3.4.14.5) by photosensitization in the presence of methylene blue at pH 7.5 was observed to have pseudo-first-order kinetics. During the process, until over 95% inactivation was achieved, the histidine and tryptophan residues were decreased from 14.0 to 2.7 and 12.6 to 7.1, respectively, per 94,000-Da subunit, without any detectable changes in other photosensitive amino acids. Modification of four histidine residues per subunit using diethylpyrocarbonate resulted in only 30% inactivation of the enzyme, while N-bromosuccinimide almost completely inactivated the enzyme with the modification of only one tryptophan residue per subunit, as determined by absorption spectrophotometry at 280 nm. The protective action of the substrate and inhibitors such as Ala-Pro-Ala and Pro-Pro against the modification of tryptophan residues with N-bromosuccinimide was observed both fluorometrically and by measurement of activity. On the basis of these results it is suggested that one of the tryptophan residues in the enzyme subunit is essential for the functioning of the substrate binding site of pig kidney dipeptidyl peptidase IV.

Chemical modification studies on Abrus agglutinin. Involvement of tryptophan residues in sugar binding.

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydrate-binding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.

A fluorescent method for selective isolation of tryptophan-containing peptide: Modification of tryptophan residue in a protein with a chelating agent, 2-carboxy-1-hydroxy-4-naphthylmethyldimethylsulfonium chloride.

A fluorescent method for selective isolation of tryptophan-containing peptide: Modification of tryptophan residue in a protein with a chelating agent, 2-carboxy-1-hydroxy-4-naphthylmethyldimethylsulfonium chloride.

Involvement of tryptophan residues in colchicine binding and the assembly of tubulin

Chemical modification of tubulin with 2-hydroxy-5-nitrobenzyl bromide, a reagent selective for tryptophan, inhibits tubulin's colchicine binding and in vitro assembly activities. Loss of colchicine binding shows a linear relationship with the modification of tryptophan residues, and is complete when not more than five residues are modified. GTP affords partial protection against this loss of colchicine binding. The in vitro assembly of tubulin is somewhat less sensitive, since microtubules are formed from tubulin dimers possessing 3–4 but not five modified residues. Furthermore, two of the eight tryptophans per dimer are reactive when tubulin is assembled into microtubules.

Effects of chemical modification of lysine, tyrosine and tryptophan residues in pea seed nucleoside diphosphate kinase and inhibition of the enzyme with antibodies.

Pea seed nucleoside diphosphate kinase (NDP kinase) is an oligomeric, tetrameric enzyme which has been shown to be phosphorylated by its substrate ATP, presumably forming an 1-phosphohistidine at its active site. Recently has also a reactive lysine residue been demonstrated at the active site. In the present investigation nitration of a tyrosine residue is shown to inactivate the enzyme. There seems only to be a slight structural change in the enzyme on modification of these essential lysine and tyrosine residues, since double diffusion experiments with an inhibitory antiserum shows no difference in reactivity between the native enzyme and the modified enzyme. It is also found that modification of all tryptophan residues in the enzyme reduces the enzyme activity only to a small degree, indication that these hydrophobic amino acid residues are not directly involved in the catalytic process.

Enzymatic modification of tryptophan residues by tryptophan side chain oxidase I and II from Pseudomonas.

The enzymatic modification of tryptophan residues in Trp-Leu, Leu-Trp, Leu-Trp-Leu, and yeast mating hormone (alpha-factor), a tridecapeptide containing Trp-1 and Trp-3, has been investigated with tryptophan side chain oxidase I crystallized previously from Pseudomonas and tryptophan side chain oxidase II recently isolated from the same organism. With Trp-Leu as substrate, threo- and erythro-beta-hydroxytryptophan residues were identified as primary products and the former perferentially underwent second step dehydrogenation to give beta-ketotryptophan. Free threo- and erythro-beta-hydroxytryptophan, hitherto undescribed tryptophan derivatives, were isolated after enzymatic hydrolysis, identified, and characterized. Leu-alpha, beta-dehydrotryptamine, a new biochemical entity, was a sole main product of Leu-Trp. With Leu-Trp-Leu, diastereoisomeric Leu-beta-hydroxyl-Trp-Leu and Leu-alpha, beta-dehydro-Trp-Leu were formed simultaneously, the pH and ionic strength being determinants of the ratio of these products. Leu-threo-beta-hydroxy-Trp-Leu was also converted to Leu-beta-keto-Trp-Leu, but, distinct from NH2-terminal beta-hydroxytryptophan, both diastereoisomers underwent acid-catalyzed dehydration to give Leu-alpha, beta-dehydro-Trp-Leu. Thus, the mode of modification was shown to be prescribed by the site of tryptophan in peptides and the results were the same with either tryptophan side chain oxidase I or II, except that the latter was almost inactive on Trp-Leu. With any substrate, 3-indolecarboxaldehyde was a sole detectable by-product.

Structure-function studies of cholera toxin and its A and B protomers. Modification of tryptophan residues.

The tryptophan residues on cholera toxin and its A and B protomers have been modified by reaction with 2-nitrophenylsulfenyl chloride and 2,4-dinitrophenylsulfenyl chloride. Modification of the tryptophan residues of cholera toxin results in complete loss of toxicity measured in a skin permeability assay. Modification of cholera toxin and its B protomer results in the complete loss of binding activity toward membrane receptors, the ganglioside galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylceramide (GM1), and the oligosaccharide moiety of the ganglioside GM1. Modification of cholera toxin and its A protomer results in a complete loss of the ADP-ribosylation activity exhibited by their native counterparts. Modification of the A protomer results in no apparent change in its physical properties by sedimentation velocity in the ultracentrifuge or by gel filtration chromatography. Modification of the B protomer, either directly or when it remains a component part of the holo toxin structure, results in a change in its sedimentation value and its elution from gel filtration columns. The changes are compatible with a conversion of the B protomer from a pentameric moiety in aqueous solvents to its existence as a monomer unit, i.e. to the individual polypeptide chains comprising the native B pentamer. Thiolysis of the 2,4-dinitrophenylsulfenyl chloride derivative of the B protomer reaggregates the individual-polypeptide chains but does not return its ability to interact with GM1.

A new reductive modification of hen's egg white lysozyme with pyridine-borane.

A reductive modification of tryptophan residues in hen's egg white lysozyme was carried out in trifluoroacetic acid with pyridine-borane. Six tryptophan residues were fully reduced to the corresponding 2, 3-dihydro compounds without any detectable change in the molecule. Enzymic activities of the reduced lysozyme was found to be completely lost.

Functional consequences of tryptophan modification in human fibrinogen

When human fibrinogen was modified with H 2O 2, inter- and intra-molecular cross-links of fibrinogen were formed, accompanied with oxidation of tryptophan, methionine and tyrosine residues. These cross-links may be closely associated with oxidation of tryptophan residues. The polymerization activity of fibrinogen with thrombin was decreased markedly by this modification. Modification of tryptophan residues in fibrinogen was also performed with 2-hydroxy-5-nitrobenzyl bromide. Modification of two out of a total 78 tryptophan residues in the molecule with the reagent led to the intensification (1.7 times) of the polymerization activity with thrombin and further modification of the next two residues led to complete loss of the polymerization activity. The first two tryptophan residues to be modified are in Fragment D, and the next two occur in Fragment E.

New procedure for oxidation of 3-substituted indoles to oxindoles: modification of tryptophan residues in peptides and proteins

A new high-yield procedure for the oxidation to oxindoles of 3-substituted indoles, including tryptophan-containing peptides and proteins, involving the use of a mixture of dimethyl sulphoxide and concentrated HCl, is described.

Hepatitis B surface antigen (HBs Ag): tryptophan content and biological activity.

SUMMARY The tryptophan content of hepatitis B surface antigen (HBsAg) was studied by spectrophotometric titration with N-bromosuccinimide (NBS) at pH 4.0 in 8.0 m-urea. A tryptophan value of 13.9 % or 22.4 residues/mol (mol. wt. = 30000) was obtained by this method. This value coupled with 2.3 mol per cent tyrosine content obtained by amino acid composition studies accounts for the unusually high extinction coefficient of HBsAg (at 280 nm E 1 cm 1 % = 37.26). The modification of tryptophan residues with NBS did not cause cleavage of the protein or loss of biologic activity as determined by radioimmunoassay.

Studies on the complement-binding site of rabbit immunoglobulin G—I. Modification of tryptophan residues and their role in anticomplementary activity of rabbit IgG

The effect of tryptophan modification of rabbit IgG and its fragment Fc on anticomplementary activity was studied using a tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl bromide. In order to locate the position of the critical tryptophan residues, modified IgG was subjected to pepsin treatment and a peptide containing modified tryptophan residues was isolated and identified as belonging to a fragment derived from Fc near the hinge region (Pep V). In order to determine if the complement-binding site of IgG could be attributed to one particular peptide from the Fc region, the interaction of normal and tryptophan-modified peptides with complement was investigated. Peptides were tested for their effect on complement fixation either directly, under specified conditions, or in the presence of aggregated IgG. In the latter case, Pep V produced an effect different from that of the other peptides. Tryptophan modification of this peptide altered its behavior implying that at least one tyrptophan residue in Pep V is involved in complement fixation by IgG. Further studies are required to localize more precisely the complement-binding site of IgG.