Photocaged variants of the MunI and PvuII restriction enzymes.

Regulation of proteins by light is a new and promising strategy for the external control of biological processes. In this study, we demonstrate the ability to regulate the catalytic activity of the MunI and PvuII restriction endonucleases with light. We used two different approaches to attach a photoremovable caging compound, 2-nitrobenzyl bromide (NBB), to functionally important regions of the two enzymes. First, we covalently attached a caging molecule at the dimer interface of MunI to generate an inactive monomer. Second, we attached NBB at the DNA binding site of the single-chain variant of PvuII (scPvuII) to prevent binding and cleavage of the DNA substrate. Upon removal of the caging group by UV irradiation, nearly 50% of the catalytic activity of MunI and 80% of the catalytic activity of PvuII could be restored.

Role of tyrosine and tryptophan in chemically modified serum albumin on its tissue distribution.

To investigate the effect of functional groups in bovine serum albumin (BSA) on its tissue distribution characteristics, tyrosine (Tyr) or tryptophan (Trp) residues of BSA were chemically modified by tetranitromethane (TNM) and 2-hydroxy-5-nitrobenzyl bromide (HNB), respectively. BSA was successfully modified with each reagent depending on the amount of the reagent added to the reaction mixture, and TNM- and HNB-modified BSA derivatives with different degrees of modification were obtained. Circular dichroism measurements showed that slight secondary and large tertiary changes were detectable as the degree of modification increased. After intravenous injection into mice, all synthetic BSA derivatives were eliminated very slowly from the systemic circulation. However, (111)In-TNM(6.6)- and (111)In-HNB(2.0)-BSA, derivatives with a high degree of modification, showed a slightly faster disappearance from the systemic circulation and slightly higher accumulation in the liver than (111)In-unmodified BSA. Pharmacokinetic analyses also demonstrated that the modification of Tyr or Trp residues on BSA had only marginal effects on tissue distribution. These results indicate that the Tyr and Trp residues have little effect on the tissue distribution characteristics of serum albumins, and that the specific modification of these residues may be a promising approach to designing sustained drug delivery systems using serum albumins.

Analysis of tryptophan surface accessibility in proteins by MALDI-TOF mass spectrometry.

Surface accessible amino acids can play an important role in proteins. They can participate in enzyme's active center structure or in specific intermolecular interactions. Thus, the information about selected amino acids' surface accessibility can contribute to the understanding of protein structure and function. In this paper, we present a simple method for surface accessibility mapping of tryptophan side chains by their chemical modification and identification by MALDI-TOF mass spectrometry. The reaction with 2-hydroxy-5-nitrobenzyl bromide, a common and highly specific covalent modification of tryptophan, seems to be very useful for this purpose. The method was tested on four model proteins with known spatial structure. In the native proteins (1) only surface accessible tryptophan side chains were found to react with the modification agent and (2) no buried one was found to react at lower reagent concentrations. These results indicate that the described method can be a potent tool for identification of surface-located tryptophan side chain in a protein of unknown conformation.

Screening major binding sites on human serum albumin by affinity capillary electrophoresis.

A screening method is described for determining whether a drug or small solute has significant interactions at the two major binding sites on human serum albumin (HSA). This method uses affinity capillary electrophoresis (ACE) to perform a mobility shift assay, where the solute of interest is injected in both the presence of pH 7.4, 0.067 M phosphate buffer, and the same buffer containing a known concentration of HSA. Dextran is also used in the running buffer to adjust the mobility of HSA. Two types of modified HSA are used in this assay. The first is modified with 2-hydroxy-5-nitrobenzyl bromide (HNB), which selectively blocks HSA's warfarin-azapropazone site. The second type of HSA is modified with tetranitromethane (TNM), which decreases binding at the indole-benzodiazepine site. By comparing the mobility of a solute in the presence of these two modified forms of HSA vs normal HSA, it is possible to detect solute interactions at these binding sites. This approach is illustrated using warfarin and ibuprofen as examples of test solutes.

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.

Identification of drug-binding sites on human serum albumin using affinity capillary electrophoresis and chemically modified proteins as buffer additives.

A technique based on affinity capillary electrophoresis (ACE) and chemically modified proteins was used to screen the binding sites of various drugs on human serum albumin (HSA). This involved using HSA as a buffer additive, following the site-selective modification of this protein at two residues (tryptophan 214 or tyrosine 411) located in its major binding regions. The migration times of four compounds (warfarin, ibuprofen, suprofen and flurbiprofen) were measured in the presence of normal or modified HSA. These times were then compared and the mobility shifts observed with the modified proteins were used to identify the binding regions of each injected solute on HSA. Items considered in optimizing this assay included the concentration of protein placed into the running buffer, the reagents used to modify HSA, and the use of dextran as a secondary additive to adjust protein mobility. The results of this method showed good agreement with those of previous reports. The advantages and disadvantages of this approach are examined, as well as its possible extension to other solutes.

Effect of protein-modifying reagents on ecto-apyrase from rat brain.

We have tested several chemical modifiers to investigate which amino acid residues, present in the primary structure of the ecto-apyrase, could be involved in catalysis. Synaptosomes from cerebral cortex of rats were prepared and the ATP diphosphohydrolase activity was assayed in absence or the presence of the modifiers. Percentages of residual activity for ATPase and ADPase obtained when the following reagents were tested, are respectively: phenylglyoxal (an arginine group modifier) 17 and 30%; Woodward's reagent (a carboxylic group modifier) 33 and 23%; Koshland's reagent (a tryptophan group modifier) 10 and 12%; maleic anhidride (an amino group modifier) 11 and 25% and carbodiimide reagent (a carboxylic group modifier) 56 and 72%. Otherwise, PMSF, a seryl protein modifier and DTNB, a SH-group modifier did not affect either ATPase or ADPase activity. Inhibitions observed after treatment with phenylglyoxal and Woodward's reagent were significantly prevented when the synaptosomal fraction was preincubated with ATP and ADP, indicating that the arginine and the side chain of glutamate or aspartate (carboxyl groups) participate in the structure of the active site. This interpretation was confirmed by using GTP and GDP, two other apyrase substrates. Phenylglyoxal and Woodward's reagent also inhibited the GTPase and GDPase activities and this inhibition was prevented by preincubation with these substrates.

Modification of the mitochondrial F1-ATPase epsilon subunit, enhancement of the ATPase activity of the IF1-F1 complex and IF1-binding dependence of the conformation of the epsilon subunit.

Treatment of bovine heart submitochondrial particles with a low concentration of 2-hydroxy-5-nitrobenzyl bromide (HNB), a selective reagent for the Trp residue of the epsilon subunit [Baracca, Barogi, Lenaz and Solaini (1993) Int. J. Biochem. 25, 1269-1275], enhances the ATP hydrolytic activity of the particles exclusively when the natural inhibitor protein IF1 is present. Similarly, isolated F1 [the catalytic sector of the mitochondrial H+-ATPase complex (ATP synthase)] treated with the reagent has the ATPase activity enhanced exclusively if IF1 is bound to it. These experiments suggest that the modification of the epsilon subunit decreases the inhibitory activity of IF1, eliciting the search for a relationship between the epsilon subunit and the inhibitory protein. Certainly, a reverse relationship exists because HNB binds covalently to the isolated F1 exclusively when the inhibitory protein is present. This finding is consistent with the existence of the epsilon subunit in different conformational states depending on whether IF1 is bound to F1 or not. Support for this assertion is obtained by measurements of the intrinsic phosphorescence decay rate of F1, a probe of the Trp epsilon subunit conformation in situ [Solaini, Baracca, Parenti-Castelli and Strambini (1993) Eur. J. Biochem. 214, 729-734]. A significant difference in phosphorescence decay rate is detected when IF1 is added to preparations of F1 previously devoid of the inhibitory protein. These studies indicate that IF1 and the epsilon subunit of the mitochondrial F1-ATPase complex are related, suggesting a possible role of the epsilon subunit in the mechanism of regulation of the mitochondrial ATP synthase.

Chemical modification of amino acid residues in glycerinated Vorticella stalk and Ca(2+)-induced contractility.

The glycerinated stalk of the peritrich ciliate Vorticella, was treated with various reagents to chemically modify the amino acid residues. The influences of these modifcations on spasmoneme contractility were investigated. First, it was confirmed that the spasmoneme contraction is not inhibited by alteration of SH groups. It was also demonstrated that chemical modification of methionine and tryptophan residues abolishes spasmoneme contractility. The reagents used for chemical modification were N-bromosuccinimide (NBS), chloramine T, and 2-hydroxy-5-nitrobenzyl bromide (HNBB), which abolished spasmoneme contractility at concentrations of 40-50 microM, 200-300 microM, and 4 mM, respectively. These results suggest that, along with Ca2+ binding proteins, there are other as yet to be identified proteins involved in contractility.

Possible mechanism for the inhibition of lectin-erythrocyte interaction in presence of endogenous lectin receptor.

The presence of hydrophobic sites in the lectin-I molecule was indicated by hydrophobic probes like 1-anilinonapthalene-8-sulfonic acid (ANS), 2-p-toluidinyl napthalene-6-sulfonic acid (TNS). N-phenyl-1-napthylamine (NA) and rose bengal (RB). This was further confirmed by amino acid modifications in the hydrophobic region of the lectin-I molecule. The binding of ANS, TNS, NA and RB to lectin-I was affected in the presence of NaCl. The involvement of hydrophobic interactions in rice-bean lectin-I-endogenous lectin receptor (ELR) complex were indicated by alterations in the circular dichroism and fluorescence emission spectra. The percentage of beta-conformation (55-63%) of lectin-I was decreased by addition of ELR. ELR on reacting with lectin-I reduced the fluorescence emissions of the hydrophobic probes while fluorescence emission of ANS, TNS, NA and RB were greatly enhanced in presence of lectin-I alone. N-aceyl-galactosamine did not change the fluorescence emissions of any of the hydrophobic probes in presence or in absence of lectin-I. This demonstrates that carbohydrate and hydrophobic sites may be different and non-interacting. It is proposed that the ELR in reacting with lectin-I, induced conformational changes in the lectin-I molecule and thereby affected its erythroagglutinating activity with human blood group "A" erythrocytes.

A conformational change associated with the phototransformation of Pisum phytochrome A as probed by fluorescence quenching.

Dynamic quenching of the two lifetime component tryptophan fluorescence of Pisum phytochrome has revealed differential accessibility of certain residues. Both acrylamide and Tl+ ions showed preferential exposure of some tryptophans in Pfr-phytochrome. Greater kq's for Pfr are, however, in contrast with values for Avena phytochrome in which Pr-->Pfr conversion impedes Tl+ access. The Pr short lifetime component was more accessible to Cs+; however, the long component accessibility was approximately 2-fold higher in Pfr. 2-Hydroxy-5-nitrobenzyl bromide (HNB-Br) modification of native Pisum phytochrome was used to reduce the total number of fluorescent tryptophans. The absence of the fluorescence contributions of the three residues which reacted with HNB-Br in both photoisomers increased the Tl+ Ksv's for Pr and Pfr. The two additional HNB-Br modifications specific for Pfr resulted in a reversal of the Stern-Volmer plots relative to the unmodified protein. The regions around four of the 10 tryptophans may represent conformationally photoresponsive areas in Pisum phytochrome A. Furthermore, topographic changes associated with the phytochrome phototransformation are not confined to the 58-kDa chromphore domain, and they involve most if not all of the region from Trp-365 to Trp-787. We also provide evidence that the protein conformation in this region is not completely conserved between Pisum and Avena phytochromes.

Interactions and effects of 2-hydroxy-5-nitrobenzyl bromide on the bovine heart mitochondrial F1-ATPase.

1. The F1-ATPase from bovine heart mitochondria was shown to chemically react and to absorb 2-hydroxy-5-nitrobenzyl bromide (HNB) with changes in catalytic properties. 2. The treatment of the enzyme with HNB at concentrations below 0.5 mM resulted in an increase of Vm and in an unchanged Km. Above 0.5 mM HNB elicited a concentration-dependent inhibition of F1. 3. HNB was found tightly bound to the enzyme epsilon-subunit whose tryptophan residue resulted modified. 4. The F1 activation appears the consequence of the covalent binding of the reagent to the enzyme, whilst inhibition results from non-covalent, reversible binding. 5. The possibility that the epsilon-subunit of mitochondrial F1-ATPase may influence the functional or regulating domain of the enzyme is discussed.

Binding of suprofen to human serum albumin. Role of the suprofen carboxyl group.

The binding of suprofen (SP), a non-steroidal anti-inflammatory drug of the arylpropionic acid class, and its methyl ester derivative (SPM) to human serum albumin (HSA) was studied by dialysis and spectroscopic techniques. In spite of the remarkable differences in the physicochemical properties of SP and SPM, the binding of each molecule to HSA was quantitatively very similar. Thermodynamic analysis suggests that the interaction of SP with HSA may be caused by electrostatic as well as hydrophobic forces, whereas the interactions with SPM may be explained by hydrophobic and van der Waals forces. Similarities in the difference UV absorption spectra between ligand-detergent micelle and -HSA systems indicate that the SP and SPM molecules are inserted into a hydrophobic crevice on HSA. The same studies suggest that the carboxyl group of SP interacts with a cationic sub-site which is closely associated with the SP binding site. Proton relaxation rate measurements indicate that the thiophen ring and propanoate portion of the SP molecule is the major binding site for HSA. The locations of SP and SPM binding sites were identified by using fluorescence probes which bind to a known site on HSA. The displacement data implied that SP primarily binds to Site II, while the high affinity site of SPM as well as low affinity site of SP are at the warfarin binding site in the Site I area. From binding data with chemically modified HSA derivatives, it is likely that highly reactive tyrosine (Tyr) and lysine (Lys) residues, which may be Tyr-411 and Lys-195, are specifically involved in SP binding. In contrast, these two residues are clearly separated from the SPM binding site. The binding of SP and SPM is independent of conformational changes on HSA that accompany N-B transition. There is evidence that the carboxyl group may play a crucial role in the high affinity binding processes of SP to HSA.

Chemical modification and 1H-NMR studies on the receptor-binding region of human interleukin 6.

Oxidation of the Met residues of human interleukin 6 (IL-6) molecule has been performed. Reactivity of Met for the oxidation reaction was found to decrease in the order of Met50, Met118, Met185, Met162, and Met68. Chemical modifications involving oxidation and carboxypeptidase A digestion of IL-6 have led to the assignments of the methyl proton resonances of Met162 and Met185, respectively. The hydroxynitrobenzyl chromophore attached to Trp158 in the IL-6 molecule showed a different absorption spectrum when the labeled IL-6 was bound to the soluble IL-6 receptor. This result indicates that Trp158 is near the receptor-binding region in IL-6. On the basis of the 1H-NMR and chemical modification data, it has been concluded that Trp158 is in spatial proximity to Met162, His165 and Met185. The receptor-binding activity decreased with an increase in the number of oxidized Met residues. Of these five Met residues, Met162 was the residue in which the receptor-binding activity decreased in the most parallel degree with that of the oxidation reaction.

Accessibility of tryptophan residues in immunoglobulin M as an index of its conformational changeability.

Demonstrated herein is the possibility of using the accessibility of tryptophan (Trp) residues in immunoglobulin M (IgM) upon modification with Koshland reagent (2-hydroxy-5-nitrobenzyl bromide) as an index of the conformational changeability of IgM. Of fourteen Trp's in the native IgM (per HL-region) only one appeared to be most accessible, evidently Trp312 in the mu-chain. Irreversible acidic and thermal conformational transitions in IgM increase the number of accessible Trp's approximately two-fold. Following partial enzymatic deglycosylation of IgM, deep scission of mannose in particular, all Trp's become inaccessible. Modification of the most accessible Trp increases 2-3 fold the number of tyrosine residues readily accessible upon nitration with tetranitromethane. Modification of four trp's using spin-label method data causes a sharp reduction of the mobility of the C mu 3 domain and a simultaneous decrease in the solubility of modified IgM.

Does 2-hydroxy-5-nitrobenzyl bromide react with the epsilon-subunit of the mitochondrial F1-ATPase?

The incubation of bovine mitochondrial F1-ATPase with 2-hydroxy-5-nitrobenzyl bromide (HNB), a selective reagent toward tryptophan residues in proteins, produced a concentration dependent inactivation of the enzyme and the covalent binding of 0.88 mol reagent/mol F1. Although HNB is highly specific for tryptophan it has also some reactivity toward cysteine, then a pre-treatment of F1 with several sulphydryl reagents has been performed to make the site of reaction clearer. This pre-treatment had neither effects in the binding stoichiometry nor in the extent of catalytic inhibition, suggesting that readly accessible thiol groups are not involved in the reaction with HNB. Since the only tryptophan bearing polypeptide of the bovine mitochondrial F1-ATPase complex is its smallest subunit, subunit-epsilon, this is the most probable candidate for HNB reaction. Therefore it may be inferred that the intactness and/or the correct conformation of this subunit could be important factor(s) for the multisite ATP hydrolytic activity of the enzyme.

Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site.

Extracellular xylanase produced in submerged culture by a thermotolerant Streptomyces T7 growing at 37-50 degrees C was purified to homogeneity by chromatography on DEAE-cellulose and gel filtration on Sephadex G-50. The purified enzyme has an Mr of 20,463 and a pI of 7.8. The pH and temperature optima for the activity were 4.5-5.5 and 60 degrees C respectively. The enzyme retained 100% of its original activity on incubation at pH 5.0 for 6 days at 50 degrees C and for 11 days at 37 degrees C. The Km and Vmax. values, as determined with soluble larch-wood xylan, were 10 mg/ml and 7.6 x 10(3) mumol/min per mg of enzyme respectively. The xylanase was devoid of cellulase activity. It was completely inhibited by Hg2+ (2 x 10(-6) M). The enzyme degraded xylan, producing xylobiose, xylo-oligosaccharides and a small amount of xylose as end products, indicating that it is an endoxylanase. Chemical modification of xylanase with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-hydroxymercuribenzoate (PHMB) revealed that 1 mol each of tryptophan and cysteine per mol of enzyme were essential for the activity. Xylan completely protected the enzyme from inactivation by the above reagents, suggesting the presence of tryptophan and cysteine at the substrate-binding site. Inactivation of xylanase by PHMB could be restored by cysteine.

Identification of amino acid residues essential for the enzymatic activities of pertussis toxin.

The enzymatic ADP-ribosyltransferase activity associated with the S1 subunit of pertussis toxin is considered to be responsible for its biological effects. Although pertussis toxin has no significant homology to other ADP-ribosylating toxins such as diphtheria toxin and Pseudomonas aeruginosa exotoxin A, the results presented in this paper show that, as for diphtheria toxin and exotoxin A, tryptophan and glutamic acid residues are essential for the enzymatic activities of pertussis toxin. Moreover, a structural motif can be identified around the critical glutamic acid residue. Chemical modification or site-directed deletion or replacement of Trp-26 abolishes ADP-ribosyltransferase and the associated NAD glycohydrolase activities. Both enzymatic activities are also abolished when Glu-129 is deleted or replaced by aspartic acid. Mutations at the Glu-106 position do not significantly reduce the enzymatic activities of the S1 subunit. The mutations do not affect the ability of the different S1 forms to be recognized by a variety of monoclonal antibodies, including neutralizing antibodies. Pertussis toxin containing a deletion or replacement of Trp-26, Glu-129, or both in the S1 subunit should thus be devoid of toxic activities without losing its reactivity with protective antibodies and, therefore, could be safely included in new generation vaccines against whooping cough.

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.

Effect of 2-hydroxy-5-nitrobenzyl bromide on proton translocation by the mitochondrial H+-ATPase.

2-Hydroxy-5-nitrobenzyl bromide, a highly reactive reagent towards tryptophan residues in proteins, is shown to activate the passive proton flux through the inner mitochondrial membrane of bovine heart submitochondrial particles (ETPH). When added at low concentrations, the reagent increased both the ATPase activity of the particles and the passive proton transport rate through the membrane. The presence of oligomycin reduced the extent of the 2-Hydroxy-5-nitrobenzyl bromide action on the proton conductivity suggesting that it acted primarily on the H+-ATPase complex. Similar effects were observed on F1-depleted particles, whilst no effect was observed on the isolated F1-ATPase activity. The results suggest that polypeptides bearing tryptophan residues may be involved in the gating function of proton channels of the mitochondrial membrane and this is particularly evident for the F0F1-ATPase complex.