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.     

Studies on the lysine-binding sites of human plasminogen. The effect of ligand structure on the binding of lysine analogs to plasminogen

A method is described for measuring relative binding constants of lysine and analogs of lysine to plasminogen and plasminogen 'kringle' fragments. Plasminogen or kringle fragments adsorbed to lysine-Sepharose are eluted with increasing concentrations of lysine or other ligands, the concentration of ligand required to elute 50% of the protein being taken as a measure of the binding constant. The method is simple and is not dependent on monitoring conformational changes. We confirm earlier reports that the best ligands for the lysine binding sites of plasminogen are omega-amino acids containing five or six carbons. We show further that both Glu-plasminogen (the native form with N-terminal glutamic acid) and Lys-plasminogen (a degraded form with N-terminal lysine), as well as the heavy chain fragments, kringle 4 and kringle 1+2+3, have very similar properties with regard to binding specificity for omega-amino acids. For all species optimal binding is observed when the distance between the amino and carboxyl carbon is about 0.68 nm. The finding of ligands is decreased by the presence of polar atoms on the alpha and beta positions of the carbon chain of amino acids. Arginine binds relatively weakly at the lysine site and there does not appear to be a separate arginine binding site in plasminogen.     

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.     

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.     

The modification of the lone tryptophan residue in human serum albumin by 2-hydroxy-5-nitrobenzyl bromide. Characterization of the modified protein and the binding of L-tryptophan and benzodiazepines to the tryptophan-modified albumin.

The possible function of the lone tryptophan residue of human serum albumin in the stereospecific binding site for indole and benzodiazepine compounds was investigated by chemical modification. This residue can be selectively modified with 2-hydroxy-5-nitrobenzyl bromide. The modification alters the conformation of the albumin only slightly, as revealed by circular dichroism, fluorescence, and ultraviolet absorption measurements. A decrease in the association constants of L-tryptophan and diazepam of about 30 - 50% and a decrease in the extrinsic Cotton effects of four benzodiazepine derivatives of about 10 - 15% were found as specific effects of the tryptophan modification. The tryptophan modification itself did not change the number of binding sites of diazepam and L-tryptophan. It is suggested that the lone tryptophan residue of human serum albumin is not directly involved in the specific binding site for indole and benzodiazepine compounds. However, the modification alters the properties of the binding site either by an incomplete refolding of the albumin after urea treatment, or a more selective allosteric effect of the modified tryptophan residue.     

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.     

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.     

Effects of monoclonal antibodies on tissue-type plasminogen activator (t-PA) binding to lysine, fibrin and heparin and on fibrin-mediated enhancement of one-chain t-PA amidolytic activity

The effects of 4 monoclonal antibodies against human tissue-type plasminogen activator (t-PA) on binding of t-PA to lysine, fibrin, and heparin, and on fibrin-mediated activation of one-chain t-PA-amidolytic activity were investigated. The association constants of the antibodies were determined in a direct assay to be equal to 0.125 l/nmol, 0.225 l/nmol, 0.4 l/nmol, and 0.5 l/nmol for mAB 5, mAB 16, mAB 25, and mAB 31, respectively. All 4 monoclonal antibodies inhibited binding of intact t-PA to lysine-Sepharose and fibrin, and they suppressed fibrin-mediated activation of one-chain t-PA-amidolytic activity. Binding analysis demonstrated that mAB 25 inhibited t-PA binding to lysine-Sepharose and to fibrin as well as fibrin-mediated enhancement of one-chain t-PA-amidolytic activity in a competitive manner with inhibitor constants of 5 nmol/l, 3 nmol/l and 10 nmol/l, respectively. It was also shown that free lysine counteracts the association of t-PA with the antibodies. Binding of t-PA to heparin is only moderately affected by the 4 antibodies. Since t-PA possesses two homologous kringle domains which contain fibrin (lysine) binding sites, the results underline the importance of a lysine binding site for fibrin binding by intact t-PA and show that the binding of the enzyme to fibrin and lysine is mediated by the same binding site of a kringle domain. The parallel effects of antibodies on fibrin binding and on fibrin-mediated enhancement of one-chain t-PA amidolytic activity proves that the site of fibrin binding is identical with the site of fibrin activation. The binding site of heparin apparently differs from lysine and fibrin binding sites.     

Kringle-kringle interactions in multimer kringle structures.

The crystal structure of a monoclinic form of human plasminogen kringle 4 (PGK4) has been solved by molecular replacement using the orthorthombic structure as a model and it has been refined by restrained least-squares methods to an R factor of 16.4% at 2.25 A resolution. The X-PLOR structure of kringle 2 of tissue plasminogen activator (t-PAK2) has been refined further using PROFFT (R = 14.5% at 2.38 A resolution). The PGK4 structure has 2 and t-PAK2 has 3 independent molecules in the asymmetric unit. There are 5 different noncrystallographic symmetry "dimers" in PGK4. Three make extensive kringle-kringle interactions related by noncrystallographic 2(1) screw axes without blocking the lysine binding site. Such associations may occur in multikringle structures such as prothrombin, hepatocyte growth factor, plasminogen (PG), and apolipoprotein [a]. The t-PAK2 structure also has noncrystallographic screw symmetry (3(1)) and mimics fibrin binding mode by having lysine of one molecule interacting electrostatically with the lysine binding site of another kringle. This ligand-like binding interaction may be important in kringle-kringle interactions involving non-lysine binding kringles with lysine or pseudo-lysine binding sites. Electrostatic intermolecular interactions involving the lysine binding site are also found in the crystal structures of PGK1 and orthorhombic PGK4. Anions associate with the cationic centers of these and t-PAK2 that appear to be more than occasional components of lysine binding site regions.     

Crystal structure of the kringle 2 domain of tissue plasminogen activator at 2.4-A resolution.

The crystal structure of the kringle 2 domain of tissue plasminogen activator was determined and refined at a resolution of 2.43 A. The overall fold of the molecule is similar to that of prothrombin kringle 1 and plasminogen kringle 4; however, there are differences in the lysine binding pocket, and two looping regions, which include insertions in kringle 2, take on very different conformations. Based on a comparison of the overall structural homology between kringle 2 and kringle 4, a new sequence alignment for kringle domains is proposed that results in a division of kringle domains into two groups, consistent with their proposed evolutionary relation. The crystal structure shows a strong interaction between a lysine residue of one molecule and the lysine/fibrin binding pocket of a noncrystallographically related neighbor. This interaction represents a good model of a bound protein ligand and is the first such ligand that has been observed in a kringle binding pocket. The structure shows an intricate network of interactions both among the binding pocket residues and between binding pocket residues and the lysine ligand. A lysine side chain is identified as the positively charged group positioned to interact with the carboxylate of lysine and lysine analogue ligands. In addition, a chloride ion is located in the kringle-kringle interface and contributes to the observed interaction between kringle molecules.     

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.     

Avoidance of strongly chaotropic eluents for immunoaffinity chromatography by chemical modification of immobilized ligand.

The need for chaotropic eluents in immunoaffinity chromatography is a consequence of the high affinities of antibodies towards their antigens. This affinity is decreased and elution of antiglucagon antibodies from a column of immobilized glucagon can be achieved under mild conditions when the steric complementarity to the antibody binding site is perturbed by selective chemical modification of the hormone. The effects of reaction with 2-hydroxy-5-nitrobenzyl bromide, tetranitromethane and hydrogen peroxide have been studied. Conversely, treatment of immobilized antibodies with 2-hydroxy-5-nitrobenzyl bromide facilitates the elution of glucagon during immunoaffinity chromatography. The general implications of these results are discussed.     

Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [125I]EDP I, [125I]Glu-plasminogen, and [125I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [125I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and 1730 microM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region (EDP I, Glu-plasminogen, Lys-plasminogen, and the plasmin heavy chain) and did not react with those lacking an EDP I region [miniplasminogen, the plasmin light chain or EDP II (kringle 4)] or with tissue plasminogen activator or prothrombin, which also contain kringles. By immunoblotting analyses, a chymotryptic degradation product of Mr 20,000 was derived from EDP I that retained reactivity with the antibody. The high-affinity lysine binding site was equally available to the antibody probe in Glu- and Lys-plasminogen and also appeared to be unoccupied in the plasmin-alpha 2-antiplasmin complex. alpha 2-Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-function relationships in the interaction of alpha-thrombin with blood platelets.

Highly purified alpha-thrombin has been chemically modified in an attempt to determine which features of the molecule are important for normal platelet-thrombin interactions. Modifying agents included diisopropylphosphorofluoridate and 1-chloro-3-tosylamido-7-amino-L-2-heptanone, which modify serine and histidine, respectively, at the catalytic site, as well as N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, which modify a single tryptophan at or near the fibrinogen-binding site. Active site-directed modification did not appreciably affect the binding characteristics, but prevented platelet activation. In contrast, modification of tryptophan at the macromolecular substrate-binding site resulted in the loss of high affinity binding of thrombin to platelets, while low affinity binding was apparently unaffected. This modification altered but did not abolish the ability of thrombin to effect platelet aggregation and release of [14C]serotonin. These results suggest that residues at the catalytic site are not involved in binding and that the macromolecular substrate-binding site of alpha-thrombin participates in high affinity binding to platelets. These data are also consistent with the existence of at least two types of binding sites for thrombin on the platelet surface as well as more than one platelet-binding region on the thrombin molecule.     

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.     

Structure and action of heteronemertine polypeptide toxins: Inactivation of Cerebratulus lacteus toxin B-IV concomitant with tryptophan alkylation

Reaction of Cerebratulus lacteus toxin B-IV with 2-hydroxy-5-nitrobenzyl bromide at pH 4.5 results in modification of toxin tryptophan residues and loss of biological activity. With relatively small reagent excesses, one tryptophan per molecule is modified without major effect on toxicity. Further reaction results in modification of a second residue of tryptophan and loss of at least 95% of the toxic activity. Modification of one or both tryptophan residues is without significant effect on the secondary structure of the protein. The specificity of each phase of the reaction has been assessed by fingerprint analysis of peptides derived from toxin modified to differing extents with 2-hydroxy-5-nitrobenzyl bromide. It is thus possible to show that tryptophan-5 reacts first and tryptophan-30 only under more rigorous conditions. It thus appears that tryptophan-30 is essential for full neurotoxic activity.     

The binding of plasminogen fragments to cultured human umbilical vein endothelial cells.

Glu-plasminogen, kringle 1-5, kringle 1-3, and miniplasminogen exhibited strong binding to human umbilical vein endothelial cells (HUVEC). On the other hand, no significant binding was obtained with microplasminogen and kringle 4. Kringle 1-5 and miniplasminogen, which both contained kringle 5, specifically inhibited the binding of plasminogen to HUVEC while kringle 1-3 did not. The results implied plasminogen molecule contained at least two binding sites, with which it interacted HUVEC. The stronger binding site was located in kringle 5 and the weaker one was in kringle 1-3. Kringle 4 and the active site domain exhibited no significant binding to HUVEC. The interaction of plasminogen with HUVEC is mainly through binding site on kringle 5.     

A role for apolipoprotein(a) in protection of the low-density lipoprotein component of lipoprotein(a) from copper-mediated oxidation

Low-density lipoprotein (LDL) oxidation is stimulated by copper. Addition of a recombinant form of apolipoprotein(a) (apo(a); the distinguishing protein component of lipoprotein(a)) containing 17 plasminogen kringle IV-like domains (17K r-apo(a)) protects LDL against oxidation by copper. Protection is specific to apo(a) and is not achieved by plasminogen or serum albumin. When Cu(2+) is added to 17K r-apo(a), its intrinsic fluorescence is quenched in a concentration-dependent and saturable manner. Quenching is unchanged whether performed aerobically or anaerobically and is reversible by ethylenediaminetetraacetate, suggesting that it is due to equilibrium binding of Cu(2+) and not to oxidative destruction of tryptophan residues. The fluorescence change exhibits a sigmoid dependence on copper concentration, and time courses of quenching are complex. At copper concentrations below 10 microM there is little quenching, whereas above 10 microM quenching proceeds immediately as a double-exponential decay. The affinity and kinetics of copper binding to 17K r-apo(a) are diminished in the presence of the lysine analogue epsilon -aminocaproic acid. We propose that copper binding to the kringle domains of 17K is mediated by a His-X-His sequence that is located about 5A from the closest tryptophan residue of the lysine binding pocket. Copper binding may account for the natural resistance to copper-mediated oxidation of lipoprotein(a) relative to LDL that has been previously reported and for the protection afforded by apo(a) from copper-mediated oxidation of LDL that we describe in the present study.     

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.     

Tryptophan residue at the heparin binding site in antithrombin III

Chemical modifications have demonstrated that the ultraviolet difference spectrum produced when heparin interacts with antithrombin III is due primarily to changes in the tryptophan environment. This is based on the observation that this spectrum could be abolished by treatment of antithrombin III with dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide but not with tetranitromethane. The tryptophan-modified antithrombin III is still capable of binding to thrombin even when it has lost 85% of heparin cofactor activity. A marked decrease in reactivity of tryptophan residues is observed when modification is carried out in the presence of heparin. Evidence is presented that tryptophan is in the heparin binding site.