The modification of tryptophan in bovine thrombin.

2-Hydroxy-5-nitrobenzyl bromide, at a 100-fold molar excess, was observed to react withthrombin at pH 4.0 to give a modified enzyme which possessed 20% of the fibrinogen clotting activity and 80% of the esterase activity compared to a control preparation. Spectrophotometric analysis of the modified protein indicated that this effect on catalytic activity was associated with the incorporation of 1 mol of reagent per mol of thrombin. Amino acid analysis showed no loss of amino acids other than tryptophan. The reaction of N-bromosuccinimide with thrombin at 2-fold molar excess resulted in the modification of one tryptophan per mol of enzyme with the loss of 80% of the fibrinogen clotting activity with, as above, a considerably smaller loss of esterase activity. Oxidation of thrombin with N-bromosuccinimide decreased the extent of subsequent tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Thrombin modified with 2-hydroxy-5-nitrobenzyl bromide showed a 3-4 fold increase in Km and a decrease in V for the ester substrate. The reaction of thrombin with 2-acetoxy-5-nitrobenzyl bromide, a substrate analogue, also resulted in the inactivation of the enzyme. The data are interpreted to show the presence of a tryptophan residue at or near the enzyme's substrate binding site.     

An active center tryptophan residue in dihydrofolate reductase: chemical modification, sequence surrounding the critical residue, and structural homology considerations.

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei contains three tryptophan residues and the amino acid sequence surrounding each tryptophan has been determined. Oxidation of one of these residues by N-bromosuccinimide at pH 6.5 can be correlated with the complete loss of enzymatic activity. Following denaturation in urea, the oxidized enzyme was alkylated with dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide. Based on amino acid analyses and absorbance measurements at 410 nm, 2.2 mol of hydroxynitrobenzyl groups was incorporated per mol of protein. Presumably, hydroxynitrobenzyl adducts are formed with the two nonessential tryptophans. From the amino acid compositions of the two major thermolytic peptides containing the hydroxynitrobenzyl label and the partial sequences of two cyanogen bromide peptides containing the tryptophans, it was deduced that tryptophan-5 and tryptophan-129 were modified and, therefore, by difference, tryptophan-21 is the functional residue which becomes oxidized. The amino acid sequence surrounding tryptophan-21 is -Leu-

-Trp-His-Leu-Pro-. In reductases from four other species, this region of the sequence is highly homologous; such a conservation in this vicinity of the primary structure may indicate a functional involvement. The proline residues at positions 20 and 24 may serve to position tryptophan-21 into the appropriate configuration for optimum substrate-binding interactions.     

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.     

Functional consequences of tryptophan modification in human fibrinogen.

When human fibrinogen was modified with H2O2, 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.     

Effect of modification on physico-chemical and biological properties of haptoglobin. Reaction with N-bromusuccinimide and 2-hydroxy-5-nitrobenzyl bromide.

N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide have been used for modification of tryptophan residues in human haptoglobin (Hp) type 2-1. Modification of three exposed tryptophan residues reduced considerably both the Hp-haemoglobin interaction and binding of the antibody against the native protein. Modification of the remaining 7-8 tryptophan residues resulted in a complete loss of those properties. Antisera directed against Hp with the modified tryptophan residues appeared to be highly specific in immunological reactions.     

An essential tryptophan residue for rabbit muscle creatine kinase

The tryptophan residues in rabbit muscle creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) have been modified by dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide after reversible protection of the reactive SH groups. The modification of two tryptophan residues as measured by spectrophotometric titration leads to complete loss of enzymatic activity. Control experiments show that reversible protection of the reactive SH groups as S-sulfonates followed by reduction results in nearly quantitative recovery of enzyme activity. The presence of a 410 nm absorption maximum and the decrease in fluorescence of the modified enzyme indicate the modification of tryptophan residues. At the same time, SH determinations after reduction of the modified enzyme show that the reagent has not affected the protected SH groups. Quantitative treatment of the data (Tsou, C.-L. (1962) Sci. Sin. 11, 1535 1558) shows that among the tryptophan residues modified, one is essential for its catalytic activity. The presence of substrates partially protects the modification of tryptophan residues as well as the inactivation, suggesting that the essential tryptophan residue is situated at the active site of this enzyme.     

Studies on the biotin-binding site of streptavidin. Tryptophan residues involved in the active site.

Streptavidin, the non-glycosylated bacterial analogue of the egg-white glycoprotein avidin, was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl (Hnb) bromide. As with avidin, complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per streptavidin subunit. Tryptic peptides obtained from an Hnb-modified streptavidin preparation were fractionated by reversed-phase h.p.l.c., and three major Hnb-containing peptide fractions were isolated. Amino acid and N-terminal sequence analysis revealed that tryptophan residues 92, 108 and 120 are modified and probably comprise part of the biotin-binding site of the streptavidin molecule. Unlike avidin, the modification of lysine residues in streptavidin failed to result in complete loss of biotin-binding activity. The data imply subtle differences in the fine structure of the respective biotin-binding sites of the two proteins.     

Consequences of the modification of tryptophan-215 on the function of bovine alpha-chymotrypsin

At pH values between 4.5 and 7.0, 2-hydroxy-5-nitrobenzyl bromide reacts selectively with tryptophan-215 in bovine α-chymotrypsin as demonstrated by the isolation of peptides containing modified amino acid residues. The degree of substitution at lower pH values indicates conformational changes in the enzyme observed previously by physico-chemical methods. The substitution of the native enzyme results in the loss of esterase activity. Nevertheless 2-hydroxy-5-nitro-benzyl chymotrypsin is still able to react with diisopropylphosphofluoridate.The catalytically inactive derivatives of α-chymotrypsin, DIP, TPCK and anhydro-chymotrypsin, as well as the complex of α-chymotrypsin with basic pancreatic trypsin inhibitor, are not modified by 2-hydroxy-5-nitrobenzyl bromide under the same conditions as those used for the native enzyme.HNB-chymotrypsin and anhydro-chymotrypsin, both catalytically inactive, form stoichiometric complexes with the basic pancreatic trypsin inhibitor whereas both PMS and DIP α-chymotrypsin did not have this complexing property. From the results of this and a preceding study (Ako et al., 1972) it is concluded that the intactness of the catalytic function of ehymotrypsin is not obligatory for the binding of basic pancreatic inhibitor.     

Effects of di-isopropyl phosphorofluoridate on rat liver microsomal and lysosomal beta-glucuronidase

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.     

Tryptophan modification by 2-hydroxy-5-nitrobenzyl bromide studied by MALDI-TOF mass spectrometry

The reaction with 2-hydroxy-5-nitrobenzyl bromide (HNB) is a common covalent modification of tryptophan. It results in several products which have been described by classical physico-chemical methods. To improve the understanding of the HNB-modified tryptophan structure, we synthesized a model peptide containing one tryptophan only, modified it by HNB, and analyzed the product by MALDI-TOF mass spectrometry. Surprisingly, several multi-modified products (up to 5 HNB moieties per one tryptophan) were identified. the influence of HNB concentration and pH on the degree of modification was also analyzed. In addition, a splitting of modified tryptophan peaks in MALDI-TOF spectrum was described; most probably, this effect is a common MALDI artifact of nitro-aromatic compounds which facilitates the identification of HNB-modified tryptophan by MALDI-TOF MS significantly.     

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.     

Modification of myosin subfragment 1 tryptophans by dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide

Modification of tryptophanyl residues (Trps) of myosin subfragments 1 (S-1) was performed with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS). Under controlled conditions, pH 6 at 0 degrees C and 10-min reaction with 10-100-fold molar excess, K+(EDTA) activity was reduced down to less than half, whereas Ca2+-ATPase activity increased and acto-S-1-ATPase was not affected. The number of modified Trps (up to 2.5) agreed well with the number of 2-hydroxy-5-nitrobenzyl moieties incorporated in S-1. The thiol groups of S-1 were not affected up to 50-fold molar excess of DHNBS, thus indicating that the modification was selective for Trps. The modification of as few as one Trp caused a blue shift of the emission spectrum, accompanied by a reduction in the fluorescence quantum yield. The accessibility of Trps to the fluorescence quencher acrylamide is drastically reduced upon modification, indicating that DHNBS-reactive Trps are more "exposed" than the DHNBS-refractive ones. DHNBS modification did not seem to affect the ATP-induced tryptophan fluorescence enhancement of S-1. The effect of DHNBS modification of the intrinsic fluorescence of S-1 indicates that the modified Trps are located in a polar environment and that they may be identical with the long-lifetime Trps of Torgerson [Torgerson, P. (1984) Biochemistry 23, 3002-3007]. The most reactive Trp is located in the N-terminal 27-kDa fragment of the S-1 heavy chain. It might also be inferred from the above data that the nonexposed and ATP-perturbed Trp(s) is (are) located in the 50-kDa fragment.     

The role of tryptophan residues in heparin-antithrombin interactions

A single tryptophan residue on antithrombin has been modified with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide. This alteration led to a 500-fold reduction in the heparin-dependent acceleration of thrombin-modified antithrombin interactions, as well as a 10-fold decrease in the avidity of the modified protease inhibitor for mucopolysaccharide. Preincubation of antithrombin with the octasaccharide binding domain of heparin prior to treatment with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide was able to suppress modification of the critical tryptophan and preserve the functional capacities of the protease inhibitor. Fluorescence quenching experiments indicated that the modifiable tryptophan groups of antithrombin were exposed to the solvent environment. Based upon these data, it was proposed that the loss of “heparin cofactor” activity of antithrombin must be predominantly due to an inability of the modified protease inhibitor to undergo a conformational transition required for mucopolysaccharide-dependent “activation” of the macromolecule.     

Chemical modification of the tryptophan residue in toxin B from the venom of the Indian cobra

The single tryptophan residue in toxin B has been converted into N′-formylkynurenine by ozonization in anhydrous formic acid, and also modified by reactions with 2-hydroxy-5-nitrobenzyl (HNB) bromide and 2-nitro-4-carboxyphenylsulphenyl (NCPS) chloride. Amino acid analyses of such modified derivatives show these reactions to be specific for tryptophan without significant effect to other amino acids. Ozonized toxin B has a residual toxicity of 80 %, and other tryptophan modified toxins retain at least half the toxicity of native toxin B. Each modified derivative gave a single fused precipitin line with native toxin on immunodiffusion against antitoxin B sera. In heterologous precipitin reactions, no significant decreases in antigenic activity of the modified derivatives were observed. The tryptophan residue at position 25 may, therefore, be part of neither the active site nor the antigenic site.     

The involvement of one of the three histidine residues of cow kappa-casein in the chymosin-initiated milk clotting process.

Cow kappa-casein has been modified by photo-oxidation in the presence of rose bengal and by the chemical reagents diethyl pyrocarbonate, 2-hydroxy-5-nitro-benzyl bromide and iodoacetic acid. Photo-oxidation resulted in the destruction of histidine and tryptophan residues and all of the histidines could be ethoxy-formylated by treatment with diethyl pyrocarbonate. Both procedures caused a loss in the susceptibility of the Phe-Met linkage of kappa-casein to chymosin hydrolysis. Treatment of kappa-casein with 2-hydroxy-5-nitrobenzyl bromide and iodoacetic acid caused the loss of tryptophan and methionine residues respectively but, in both cases, the susceptibility of the modified protein to chymosin hydrolysis remained unaffected. Of the amino acids examined it is concluded that only the histidine residues of cow kappa-casein are important for the hydrolytic action of chymosin and, furthermore, the treatment with diethyl pyrocarbonate suggests that only one of the three histidines plays an essential role.     

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.     

Observations on the reaction of 2-hydroxy-5-nitrobenzyl bromide with a peptide-bound tryptophanyl residue

Previous studies on the isolation of peptides containing tryptophanyl residues modified with 2-hydroxy-5-nitrobenzyl bromide demonstrated multiple products of reaction at the same residue as well as technical difficulties in the primary structure analysis of peptides containing the modified tryptophanyl residue. The present study was undertaken to explore the reaction of 2-hydroxy-5-nitrobenzyl bromide with the single tryptophanyl residue in a synthetic peptide, experimental allergenic encephalitogenic peptide. The modification of this peptide was accomplished in sodium acetate, pH 4.75, and reagent removed by gel filtration. Amino acid analysis of the modified peptide suggested that only the tryptophanyl residue had been modified under these experimental conditions. The modified peptide could be separated into multiple derivatives by high-performance liquid chromatography. Although it is clear that some of the observed heterogeneity reflects a difference in the degree of substitution at the single tryptophanyl residue, several of the derivatives appear to have the same extent of substitution. It is suggested that the heterogeneity observed is a reflection of the establishment of a new diastereoisomeric center in the peptide. These results are consistent with previous observations from other laboratories and provide a basis for the explanation of apparent heterogeneity of peptides obtained from modified proteins.     

Accessibility of tryptophan residues in immunoglobulin M molecule as an indicator of its conformational variability

The accessibility of tryptophan residues in immunoglobulin M to modification with the Koshland reagent (2-hydroxy-5-nitrobenzyl bromide) was used as an indicator of its conformational variability. Of 14 tryptophan residues (per HL-fragment) in the native IgM, only one (presumably Trp312 in the mu-chain) was the most accessible. Irreversible acid- or temperature-induced conformational changes of IgM increased almost 2-fold the number of accessible tryptophan residues. After partial enzymatic deglycosylation of IgM (especially by an intense splitting of mannose), all tryptophan residues became inaccessible. Modification of the most accessible tryptophan residue increased 2- to 3-fold the number of tyrosine residues accessible to nitration with tetranitromethane. Using the spin label method, it was demonstrated that modification of four tryptophan residues in IgM considerably decreased the mobility of the Cmu 3 domain together with an essential drop in. the solubility of the modified IgM.     

Kinetic study on chemical modification of taka-amylase A. I. Location and role of tryptophan residues

1. Five and four tryptophan residues in Taka-amylase A [EC 3.2.1.1] of A. oryzae (TAA) were modified with dimethyl(2-hydroxy-5-nitrobenzyl)-sulfonium bromide (K-IWS) in the absence and the presence of 15% maltose (substrate analog), respectively. Only one tryptophan residue was modified with dimethyl(2-methoxy-5-nitrobenzyl)-sulfonium bromide (K-IIWS) irrespective of the presence or absence of maltose. Kinetic parameters (molecular activity, k0, Michaelis constant, Km, and inhibitor constant, Ki) of the enzyme modified with K-IWS and K-IIWS were determined. The k0 value decreased with increase in the number of modified residues, but Km and Ki values and the type of inhibition were not altered by the modification. 2. The fluorescence quenching reaction of TAA with N-bromosuccinimide (NBS) proceeded in three phases. The second-order rate constants of the three phases were determined to be (4.3 +/- 0.5) x 10(5) M-1 . s-1, (2.1 +/- 0.3) x 10(3) M-1 . s-1 and (1.7 +/- 0.2) x 10(2) M-1 . s-1, respectively. In the presence of maltose, the first phase was further separated into two phases with rate constants of (4.6 +/- 0.6) x 10(6) M-1 . s-1 and (6.9 +/- 1.1) x 10(4) M-1 . s-1, respectively. On the basis of the results, it is estimated that five out of nine tryptophan residues are accessible to the solvent and among them, two tryptophan residues are substantially exposed: one is located in the maltose binding site near the catalytic site (its modification affects the catalytic function), and the other exists on the enzyme surface far from the active site.     

On the role of tryptophan residues in the mechanism of action of glyceraldehyde-3phosphate dehydrogenase as tested by specific modification.

A method is described to selectively modify one of the three tryptophan residues of the subunit of glyceraldehyde-3-phosphate dehydrogenase from yeast. As modifying agent dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide was used. The residue which is modified by the procedure described has been identified as Trp-193. There are either one or two molecules of the modifying agent being added to this tryptophan side chain. The modification apparently does not cause a detectable conformational change of the protein as judged from the methods employed. However, the enzymatic activities in the dehydrogenase as well as in the esterase reactions are lost after the modification. It could be established that the modification rendered the enzyme unable to bind the oxidized coenzyme. Also the charge-transfer interaction between enzyme and coenzyme could no longer be observed.