Human thrombins. Group IA and IIA salt-dependent properties of alpha-thrombin.

The conformations of human alpha-thrombin and gamma-thrombin have been compared by circular dichroism, solvent perturbation different spectroscopy, and chemical modification. Circular dichroism studies indicate that proteolytic conversion of alpha-thrombin to gamma-thrombin is accompanied by considerable conformational changes which include a decrease in alpha-helical content from 5-7% to 0-1%. Solvent perturbation at pH 6.0 obtained with 20% ethylene glycol, 20% glycerol, and 20% dimethyl sulfoxide indicates an apparent exposure of 3.5 +2- 0.2 tryptophan and 7.8 +/- 0.1 tyrosine residues in alpha-thrombin and 4.6 +/- 0.2 tryptophan and 9.2 +/0 0.3 tyrosine residues in gamma-thrombin. This increased exposure is substantiated by the greater reactivity of tryptophan residues in gamma-thrombin toward dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide. It suggests that gamma-thrombin is a less compact molecule than the parent alpha-thrombin. Solvent perturbation studies of alpha-thrombin and gamma=thrombin inhibited by phenyl-methanesulfonyl fluoride showed that 0.3 +/- tryptophan and 0.9 +/- 0.3 tyrosine residues in alpha-thrombin and 0.6 +/- 0.3 tryptophan and 1.3 +/- 0.4 tyrosine residues in gamma-thrombin were blocked by the inhibitor. These subtle differences in the extent of blocking of tyrosine and tryptophan suggest a tighter conformation in the catalytic site of gamma-thrombin compared to that of alpha-thrombin.

Section: Resultscontrasting

confidence: 74%

Conformational differences between high clotting human .alpha.-thrombin and nonclotting .gamma.-thrombin

Villanueva¹

1981

“…Over the range of salt concentrations used in the present study, significant changes in the values of kal and Km for the substrate D-Phe-Pip-Arg-pNA were not observed (Table I), and these results suggest that any salt-induced changes in the active-site region of thrombin were minor. Similar conclusions regarding the effect of salt on the active site of thrombin have been drawn from ESR experiments using a spin-labeled derivative of thrombin (Landis et al, 1981). Changes in the binding energy of hirudin caused by salt effects on the structure of thrombin would not necessarily be described by eq 4a or 4b.…”

Section: Discussionmentioning

confidence: 55%

“…Such secondary salt effects are difficult to exclude. Indeed, increasing concentrations of salt have been found to cause small changes in the structure of thrombin (Landis et al, 1981;Villanueva & Ferret, 1983). Over the range of salt concentrations used in the present study, significant changes in the values of kal and Km for the substrate D-Phe-Pip-Arg-pNA were not observed (Table I), and these results suggest that any salt-induced changes in the active-site region of thrombin were minor.…”

Section: Discussionmentioning

confidence: 99%

Quantitative evaluation of the contribution of ionic interactions to the formation of the thrombin-hirudin complex

Stone

Dennis²,

Hofsteenge³

1989

129

The effect of ionic strength on the kinetics of inhibition of human alpha-thrombin has been examined by using genetically engineered forms of hirudin that differed only in the number of negatively charged residues in the carboxyl-terminal region of the molecule. Analysis of the data obtained allowed the binding energy for the thrombin-hirudin complex to be divided into contributions from ionic and nonionic interactions. The contribution of nonionic interactions to the binding energy was the same for each of the forms whereas the ionic contribution varied with the charge of the molecule. Each of the negatively charged residues made an approximately equal contribution of -4kJ mol-1 to the binding energy. For native hirudin, ionic interactions accounted for 32% of the binding energy at an ionic strength of zero.

“…In order to assess where -thrombin might predominantly interact with fibrin(ogen), the peptide of residues 17-27 of the Aa chain and the NDSK fragment of human fibrinogen were chosen as likely candidates. The synthetic substrate Bz-Arg-OEt was selected for potentiometric titration because of its relatively simple kinetic behavior and moderately low affinity for thrombin [e.g., ^(limited) and Km of ~6 µ for human -thrombin in 0.15 M NaCl], which should permit detection of moderately weak inhibitors (Landis et al, 1981). When an 11-residue synthetic peptide was examined, no inhibition was obtained even up to 10 mM concentrations.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, under the present conditions of pH 7.4 for CG-50 resin chromatography, -thrombin is predominantly a noncharged protein and should not bind to this resin if such binding were attributable to "general ion exchange effects" for evenly distributed charges on the protein. That the retention by the resin might be due to nonionic adsorption and solvation with increasing salt concentrations seems unlikely since human athrombin has a solubility of up to 0.3 g/L in deionized water (Landis et al, 1981), and by use the neutral pH absorption coefficient for the protein (Fenton et al, 1977a), concentrations of roughly one-third this can be calculated from the present peak absorbances (Figures 1 and 2). Thus, the binding of -thrombin to CG-50 resin is by uneven charge distribution, which is supported by the clustering of arginines and lysines in thrombin three-dimensional models (Bing et al, 1981;Fenton & Bing, 1986).…”

Section: Discussionmentioning

confidence: 99%

Anion-binding exosite of human .alpha.-thrombin and fibrin(ogen) recognition

Fenton

Olson²,

Zabrinski³

et al. 1988

182

103

Activation of prothrombin to alpha-thrombin generates not only the catalytic site and associated regions but also an independent site (an exosite) which binds anionic substances, such as Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)]. Like human alpha-thrombin with high fibrinogen clotting activity (peak elution at I = 0.40 +/- 0.01 M, pH 7.4, approximately 23 degrees C), catalytically inactivated forms (e.g., i-Pr2P-alpha- and D-Phe-Pro-Arg-CH2-alpha-thrombins) were eluted with only slightly lower salt concentrations (I = 0.36-0.39 M), while gamma-thrombin with very low clotting activity was eluted with much lower concentrations (I = 0.29 M) and the hirudin complex of alpha-thrombin was not retained by the resin. In a similar manner, hirudin complexes of alpha-, i-Pr2P-alpha-, and gamma-thrombin were not retained by nonpolymerized fibrin-agarose resin. Moreover, the ionic strengths for the elution from the CG-50 resin of seven thrombin forms were directly correlated with those from the fibrin resin (y = 0.15 + 0.96x, r = 0.95). In other experiments, the 17 through 27 synthetic peptide of the human fibrinogen A alpha chain was not an inhibitor of alpha-thrombin, while the NH2-terminal disulfide knot (NDSK) fragment was a simple competitive inhibitor of alpha-thrombin with a Ki approximately 3 microM (0.15 M NaCl, pH 7.3, approximately 23 degrees C). These data suggest that alpha-thrombin recognizes fibrin(ogen) by a negatively charged surface, noncontiguous with the A alpha cleavage site but found within the NDSK fragment. Such interaction involving an anion-binding exosite may explain the exceptional specificity of alpha-thrombin for the A alpha cleavage in fibrinogen and alpha-thrombin incorporation into fibrin clots.