Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to the 33,000 Mr and 54,000 Mr species of human urokinase: thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to the 33,000 Mr and 54,000 Mr species of human urokinase (EC 3.4.21.

Trypsin-like serine proteinase action: determination of the catalytic parameters KS, k+2 and k/3 under conditions where the substrate exceeds the enzyme concentration.

A method for the determination of the catalytic parameters Ks, k+2 and k+3 describing trypsin-like serine proteinase action has been developed from the quantitative analysis of the kinetics of hydrolysis of two specific chromogenic substrates, N-alpha-carbobenzoxy-L-arginine p-nitrophenyl ester and N-alpha-carbobenzoxy-L-lysine p-nitrophenyl ester, catalyzed by porcine pancreatic betta-kallikrein B, bovine betta-trypsin and human urokinase (Mr 54,000 species), under conditions where the concentration of the substrate exceeds that of the enzyme. Value of Ks, k+2 and k+3 have been estimated from the effect of substrate concentration on the apparent first-order rate constant of the time-course of the burst phase of p-nitrophenol release preceding the steady-state reaction.

Controlled proteolysis of mouse epidermal growth factor. An RP-HPLC and 1H-n.m.r. study.

Tryptic digestion of the mouse epidermal growth factor (mEGF) and the chromatographic separation of its proteolytic fragments by RP-HPLC affords the isolation of the pure hormone, of its 1-48 (Des(49-53)mEGF) and 1-45 (Des(46-53)mEGF) derivatives, and of the carboxyl-terminal pentapeptide W49-W50-E51-L52-R53. Kinetics of mEGF proteolytic degradation follows a two-state time-course: native mEGF being converted into Des(49-53)mEGF with an apparent half-time of 10 min; and Des(49-53)mEGF subsequently hydrolyzed to Des(46-53)mEGF with an apparent half-time of 7 h. Native mEGF and its proteolytic fragments have been characterized by 1H-n.

Preliminary crystallographic data on the complex of bovine alpha-chymotrypsin with the recombinant proteinase inhibitor eglin c from Hirudo medicinalis.

The molecular complex built by bovine alpha-chymotrypsin and the recombinant proteinase inhibitor eglin c from Hirudo medicinalis has been crystallized from polyethylene glycol solutions, using a twofold molar excess of the inhibitor with respect to the serine proteinase. The optimum pH for crystal growth is 6.5.

Binding of inositol hexakisphosphate to the oxygenated derivative of dromedary (Camelus dromedarius) and human hemoglobin: 31P-NMR study.

Binding of inositol hexakisphosphate (IHP) to the oxygenated derivative of dromedary (Camelus dromedarius) and human hemoglobin (Hb) was investigated by 31P-NMR. The results obtained show that dromedary Hb binds, with different affinity, two IHP molecules per tetramer at distinct sites, while human Hb binds only one IHP molecule per tetramer.

pH effects on the haem iron co-ordination state in the nitric oxide and deoxy derivatives of ferrous horseradish peroxidase and cytochrome c peroxidase.

The spectral (e.p.r.

Aplysia limacina myoglobin. Crystallographic analysis at 1.6 A resolution.

The crystal structure of the ferric form of myoglobin from the mollusc Aplysia limacina has been refined at 1.6 A resolution, by restrained crystallographic refinement methods. The crystallographic R-factor is 0.

Inhibition of serine proteinases by p-carbethoxyphenyl esters of epsilon-guanidino- and epsilon-amino caproic acid: thermodynamic and molecular modeling study.

The inhibitory effect of the clinically used p-carbethoxyphenyl ester of epsilon-guanidino-caproic acid methanesulphonate (epsilon-GCA-CEP) on the catalytic properties of human LYS77-plasmin (EC 3.4.21.

Kinetic evidence for a role of heme geometry on the modulation of carbon monoxide reactivity in human hemoglobin.

Kinetics of CO binding to human hemoglobin (Hb) has been followed below neutrality. With respect to the behavior observed at pH 7.0, CO binding to deoxy-Hb at pH 2.

A comparative graphical method for the characterization of all types of linear enzyme inhibition.

A simple graphical method is described for plotting kinetics for inhibited enzyme-catalyzed reactions. The plots are particularly designed for the characterization of all types of linear enzyme inhibition in comparative studies.

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human alpha-, beta- and gamma-thrombin; a kinetic and thermodynamic study.

Kinetic and thermodynamic parameters for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human alpha-, beta- and gamma-thrombin have been determined, between 5 and 45 degrees C, at pH 7.5. BPTI-binding properties to human thrombins have been analyzed in parallel with those of serine (pro)enzymes acting on cationic and non-cationic substrates, with particular reference to the bovine beta-trypsin/BPTI system.

Effect of inositol hexakisphosphate on the spectroscopic properties of the nitric oxide derivative of ferrous naturally glycated human hemoglobin HbA1c.

The effect of inositol hexakisphosphate (IHP) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous naturally glycated human hemoglobin HbA1c (HbA1cNO) has been investigated quantitatively. The results obtained show that 1) both in the absence and presence of IHP, the EPR and absorbance spectra of HbA1cNO show the same basic characteristics described for the nitrosyl derivative of ferrous HbA0, the nonglycated major component of human hemoglobin (HbA0NO); and 2) HbA1cNO binds IHP with an apparent dissociation equilibrium constant (upsilon = 1.8 x 10(-2) M), which is at least four orders of magnitude higher than that estimated for the polyphosphate interaction with HbA0NO (less than or equal to 3 x 10(-6) M).

Zymogen activation: effect of peptides sequentially related to the bovine beta-trypsin N-terminus on Kazal inhibitor and benzamidine binding to bovine trypsinogen.

The activating effect of peptides sequentially related to the Ile 16-Val17-Gly18 N-terminus of bovine beta-trypsin (namely Ile-Val-Gly, Ile-Val, Ile-Leu, Ile-Ala, Val-Val, Leu-Val, and Val-Leu) on the thermodynamic parameters for the binding of the porcine pancreatic secretory trypsin inhibitor (Kazal inhibitor) and benzamidine to bovine trypsinogen was investigated at pH 5.5 (Bis tris-HCl buffer, I = 0.1 M) and T = 21 +/- 0.

Effect of polyanions on the spectroscopic properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin.

The effect of inositol hexakisphosphate, 2,3-diphosphoglycerate, dextran sulphate, and heparin on the spectroscopic (absorbance, circular dichroism, EPR) properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin was investigated. The results obtained show that: (i) all polyanions bind to the protein at the same sites, but with different affinities; (ii) polyanions affect the protein conformation of the ferrous nitrosyl derivative in a different way with respect to aquo-ferric and ferrous oxy dromedary hemoglobin; and (iii) the data obtained provide further independent evidence for the existence in dromedary hemoglobin of two functionally distinct polyanion binding sites that affect the conformational equilibrium of the protein in opposite ways.

Aromatic amidines: inhibitory effect on purified plasma serine proteinases, blood coagulation and platelet aggregation.

The inhibitory effect of benzamidine as well as of 1,3-di-(p-amidinophenoxy)-2,2-bis-(p-amidinophenoxymethyl)propane (TAPP-H) and TAPP-halo derivatives (with Cl, Br or I) on (i) the catalytic properties of purified plasma serine proteinases (notably, factor Xa, factor VIIa, thrombin and plasmin), (ii) blood coagulation, and (iii) platelet aggregation was investigated in vitro. For all the enzyme/inhibitor systems examined, the inhibition patterns were strictly competitive, and titrations conformed to simple equilibria. The inhibitory effect of TAPP-H and TAPP-halo derivatives is higher, by at least 10-fold, than that of benzamidine, which binds at the primary specificity subsite (S1) of serine proteinases and is commonly taken as a molecular inhibitor model.

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase, bovine alpha-chymotrypsin and subtilisin Carlsberg: thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase (EC 3.4.21.

Binding of the bovine pancreatic secretory trypsin inhibitor (Kazal) to bovine serine (pro)enzymes.

The effect of temperature and pH on the association equilibrium constant (Ka) for the binding of the bovine pancreatic secretory trypsin inhibitor (bovine PSTI, type I; Kazal inhibitor) to bovine beta-trypsin, bovine alpha-chymotrypsin and bovine trypsinogen has been investigated. The results suggest that serine (pro)enzyme inhibitor interaction involves both rigorous spatial configuration and molecular flexibility.

Active-site titration of serine proteinases acting selectively on cationic substrates by N-alpha-carbobenzoxy-L-arginine p-nitrophenyl ester and N-alpha-carbobenzoxy-L-lysine p-nitrophenyl ester; determination of active enzyme concentration.

A titration method for determination of trypsin-like serine proteinase concentration has been developed by using ZArgONp and ZLysONp, two specific chromogenic amino-acid derivatives which show the characteristics of optimal active-site titrants. Active proteinase concentration has been estimated from the effect of titrant concentration on the amplitude, at time zero, of the time-course for the instantaneous release of p-nitrophenol, preceding the steady-state reaction (burst phase).

Binding of the Ile-Val and Val-Val effector dipeptides to the binary adducts of bovine trypsinogen with Kunitz and Kazal inhibitors as well as the acylating agent p-nitrophenyl p-guanidinobenzoate. A thermodynamic and kinetic study.

Thermodynamics and kinetics of binding of the Ile-Val and Val-Val effector dipeptides to the binary adducts of bovine trypsinogen with the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor), the porcine pancreatic secretory inhibitor (PSTI, Kazal inhibitor) and the acylating agent p-nitrophenyl p-guanidinobenzoate have been investigated at pH 7.4 and 21(+/- 0.5) degrees C.

The pH dependence of pre-steady-state and steady-state kinetics for the papain-catalyzed hydrolysis of N-alpha-carbobenzoxyglycine p-nitrophenyl ester.

Pre-steady-state and steady-state kinetics of the papain (EC 3.4.22.

Fluorescence and energy transfer of tryptophans in Aplysia myoglobin.

The fluorescence decay of tryptophan residues in apo and met Aplysia limacina myoglobin and sperm whale myoglobin were measured in aqueous solution at 10 degrees-15 degrees C. In all species, multiexponential behavior was observed in which the individual components displayed unique frequency-dependent emission characteristics. The results suggest that the tryptophan fluorescence in all met samples are quenched by rapid Forster energy transfer to the heme as predicted from the crystal geometry.

Effect of inositol hexakisphosphate on the EPR properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin. Evidence for two polyanion binding sites.

The effect of inositol hexakisphosphate on the EPR properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin has been investigated at 110 K. In the absence of inositol hexakisphosphate, the nitrosyl derivative of dromedary hemoglobin shows an EPR spectrum with a rhombic shape and a weak hyperfine splitting in the gz region, a feature that is generally taken as characteristic of the high-affinity state of tetrameric hemoproteins. On addition of 1 mole of inositol hexakisphosphate/tetramer, three new hyperfine lines (Az = 1.

Binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds to bovine beta-trypsin: thermodynamic study.

The effect of pH and temperature on the association equilibrium constant (Ka) for the binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds (MTI) to bovine beta-trypsin (EC 3.4.

Inhibition of serine proteinases by tetra-p-amidinophenoxy-neo-pentane: thermodynamic and molecular modeling study.

The inhibitory effect of the aromatic tetra-benzamidine derivative tetra-p-amidinophenoxy-neo-pentane (TAPP) on the catalytic properties of beta-trypsin (EC 3.4.21.