Fluorescence stopped flow analysis of the interaction of virginiamycin components and erythromycin with bacterial ribosomes

The kinetics of the interaction between the 50 S subunits (R) of bacterial ribosomes and the antibiotics virginiamycin S (VS), virginiamycin M (VM), and erythromycin have been studied by stopped flow fluorimetric analysis, based on the enhancement of VS fluorescence upon its binding to the 50 S ribosomal subunit. Virginiamycin components M and S exhibit a synergistic effect in vivo, which is characterized in vitro by a 5- to 10-fold increase of the affinity of ribosomes for VS, and by the loss of the ability of erythromycin to displace VS subsequent to the conformational change (from R to R*) produced by transient contact of ribosomes with VM. Our kinetic studies show that the VM-induced increase of the ribosomal affinity for VS (K*VS = 25 X 10(6) M-1 instead of KVS = 5.5 X 10(6) M-1) is due to a decrease of the dissociation rate constant (k*-VS = 0.008 s-1 instead of 0.04 s-1). The association rate constant remains practically the same (k+VS approximately k*+VS = 2.8 X 10(5) M-1 s-1), irrespective of the presence of VM. VS and erythromycin bind competitively to ribosomes. This effect has been exploited to determine the dissociation rate constant of VS directly by displacement experiments from VS . 50 S complexes, and the association rate constant of erythromycin (k+Ery = 3.2 X 10(5) M-1 S-1) on the basis of competition experiments for binding of free erythromycin and VS to ribosomes. By making use of the change in competition behavior of erythromycin and VS, after interaction of ribosomes with VM, the conformational change induced by VM has been explored. Within the experimentally available concentration region, the catalytic effect of VM has been shown to be coupled to its binding kinetics, and the association rate constant of VM has been determined (k+VM = 1.4 X 10(4) M-1 S-1). Evidence is presented for a low affinity binding of erythromycin (K*Ery approximately 3.3 X 10(4) M-1) to ribosomes altered by contact with VM. A model involving a sequence of 5 reactions has been proposed to explain the replacement of ribosome-bound erythromycin by VS upon contact of 50 S subunits with VM.     

Oxygen binding to dithionite-reduced chloroperoxidase

Both the kinetics of ferric chloroperoxidase reduction by dithionite and the binding of molecular oxygen to ferrous chloroperoxidase have been studied. The oxyferrous chloroperoxidase decays spontaneously to the ferric enzyme. In addition the corresponding rapid-scan spectra have been recorded. The reduction reaction is caused by SO-.2 with a rate constant of (7.7 +/- 1.0) X 10(4) M-1 S-1. Oxygen binding occurs with a rate constant of (5.5 +/- 1.0) X 10(5) M-1 S-1 over the pH range 3.5-6. Oxyferrous chloroperoxidase has a Soret absorption peak at 428 nm and two partially resolved peaks at 555 nm and 588 nm. Isosbestic points occur at the following wavelengths: between ferrous and oxyferrous chloroperoxidase at 419, 545, 555 and 580 nm; between oxyferrous and ferric chloroperoxidase at 419, 487, 540, 609 and 682 nm.     

Action mechanism of Escherichia coli DNA photolyase. I. Formation of the enzyme-substrate complex

Escherichia coli DNA photolyase (photoreactivating enzyme) is a flavoprotein. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and, upon illumination with 300-600 nm radiation, catalyzes the photosensitized cleavage of the cyclobutane ring thus restoring the integrity of the DNA. We have studied the binding reaction using the techniques of nitrocellulose filter binding and flash photolysis. The enzyme binds to dimer-containing DNA with an association rate constant k1 estimated by two different methods to be 1.4 X 10(6) to 4.2 X 10(6) M-1 S-1. The dissociation of the enzyme from dimer-containing DNA displays biphasic kinetics; for the rapidly dissociating class of complexes k2 = 2-3 X 10(-2) S-1, while for the more slowly dissociating class k2 = 1.3 X 10(-3) to 6 X 10(-4) S-1. The equilibrium association constant KA, as determined by the nitrocellulose filter binding assay and the flash photolysis assay, was 4.7 X 10(7) to 6 X 10(7) M-1, in reasonable agreement with the values predicted from k1 and k2. From the dependence of the association constant on ionic strength we conclude that the enzyme contacts no more than two phosphodiester bonds upon binding; this strongly suggests that the pyrimidine dimer is the main structural determinant of specific photolyase-DNA interaction and that nonspecific ionic interactions do not contribute significantly to substrate binding.     

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

Effects of the ligands of beef tryptophanyl-tRNA synthetase on the elementary steps of the tRNA(Trp) aminoacylation

Tryptophanyl-tRNA synthetase catalyzed formation of Trp-tRNA(Trp) has been studied by mixing tRNA(Trp) with a preformed bis(tryptophanyl adenylate)-enzyme complex in the 0-60-ms time range, on a quenched-flow apparatus. Analyzing the data gives an association rate constant ka = (1.22 +/- 0.47) X 10(8) M-1 S-1, a dissociation rate constant kd = 143 +/- 73 S-1, and a dissociation constant Kd = 1.34 +/- 0.80 microM for tRNA(Trp). The maximum rate constant of tryptophan transfer to tRNA(Trp) is kt = 33 +/- 3 S-1. When starting the aminoacylation reaction with a mono(tryptophanyl adenylate)-enzyme complex, one obtains different kinetic profiles than when using a bis(tryptophanyl adenylate)-enzyme complex. Over a 0-400-ms time range, the monoadenylate-enzyme complex yields an apparent first-order reaction, while the bis-adenylate-enzyme complex yields a biphasic aminoacylation of tRNA(Trp). Analysis of Trp-tRNA(Trp) formation from both complexes according to simple reaction schemes shows that the dissociation of tRNA(Trp) from an enzyme subunit carrying no adenylate is 6.9-fold slower than from an enzyme subunit carrying an adenylate. The apparent rate constant of dissociation of nascent tryptophanyl-tRNA(Trp) is 4.9 S-1 in the absence of free tryptophan, which is much slower than its rate of formation (33 S-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Quenching of the zinc-protoporphyrin triplet state as a measure of small-molecule diffusion through the structure of myoglobin

The diffusion of small molecules through the myoglobin structure was studied. It has been shown that the fluorescent Zn-protoporphyrin substitutes easily for the native nonfluorescent Fe-protoporphyrin in myoglobin. The quenching rate of the E-type delayed fluorescence of Zn-protoporphyrin in a substituted myoglobin by the quenchers oxygen and anthraquinonesulfonate was used to measure their diffusion from the ambient solution through the protein to the ligand binding site. The quenching rate constant (at 21 degrees C) for oxygen is kq = (9.6 +/- 0.9) X 10(7) M-1 S-1, only 1 order of magnitude less than that for Zn-hematoporphyrin quenching in aqueous solution. The activation energy in the range between 2 and 40 degrees C is Ea = 6.0 +/- 0.6 kcal/mol. The corresponding data for anthraquinonesulfonate are kq = (2.1 +/- 0.3) X 10(8) M-1 S-1 and Ea = 5.8 +/- 0.6 kcal/mol. Taking into account the statistical factor involved in the oxygen quenching of the Zn-porphyrin triplet, the quenching rates are very similar. The data are discussed in terms of the "gated reaction" theory of Northrup and McCammon. The similar rate constants and activation energies indicate that the diffusion rate in the protein is determined by the frequency of the conformational changes that open "gates" for the passage of the quencher through the protein.     

Kinetics and mechanism of bilirubin binding to human serum albumin

The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.     

Bovine cardiac myosin subfragment 1. Transient kinetics of ATP hydrolysis

The kinetics of binding and hydrolysis of ATP by bovine cardiac myosin subfragment 1 has been reinvestigated. More than 90% of the total fluorescence amplitude associated with ATP hydrolysis occurs with an apparent second-order rate constant of 8.1 X 10(5) M-1 S-1 and a limiting rate constant of approximately 140 S-1 (100 mM KCl, 50 mM 1,3-bis-[tris(hydroxymethyl)methylamino]-propane, 10 mM MgCl2, pH 7.0, 20 degrees C); the remaining 10% occurs more slowly (approximately 1 S-1). The observed rate constants are independent of subfragment 1 concentration under pseudo first-order conditions for ATP with respect to protein. The fraction of protein which hydrolyzes ATP rapidly is not a function of the nucleotide or protein concentration and appears to be constant irrespective of ionic strength or temperature within the range studied (50-100 mM KCl, pH 7.0, 15-20 degrees C). These data are compared to that obtained previously using subfragment 1 prepared by a different method which showed ATP-dependent aggregation of two protein species.     

Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart. Kinetics of thiamine diphosphate binding to the complex

In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.     

Kinetics of CO binding to and dissociation from microsomal cytochrome P-450 induced by phenobarbital in rat liver

The kinetics of CO binding to hepatic microsomes from phenobarbital-treated Sprague-Dawley rats, measured by stopped flow spectrophotometry, can be resolved into three components with second order velocity constants of 2.23 +/- 0.35 X 10(5) M-1 S-1, 1.59 +/- 0.18 X 10(6) M-1 S-1, and 8.7 +/- 1.7 X 10(6) M-1 S-1. The three CO-binding species were present in ratios of 1:1.25:1.39 as judged by the relative amplitude of the change in absorbance at 450 nm associated with each of the kinetic components. Similar results were obtained in a range of [CO] from 10 to 700 micron when CO recombination was followed subsequent to flash photolysis of the CO-associated microsomes. In contrast, the dissociation rate of CO from its cytochrome P-450 complex measured by the NO replacement method was biphasic. Approximately 40% of the bound CO dissociated at a rate of 0.40 +/- 0.071 s-1, whereas the remaining 60% dissociated at a rate of 0.049 +/- 0.008 s-1 at 20 degrees C.     

Occurrence and properties of a prostaglandin F2alpha receptor in bovine corpora lutea.

Prostaglandin F2alpha was specifically bound by a particulate fraction from bovine corpora lutea. The rate constants for the association (7.5 X 10(3) M-1 S-1) and dissociation (2.1 X 10-4 S-1) reactions gave a dissociation constant of 2.8 X 10(-8) M which is similar to that determined from a Scatchard plot of binding data at equilibrium (5 X 10(-8) M). The receptor was stable for several hours at 23 degrees C but was rapidly destroyed at 37 degrees C. The pH optimum for the binding reaction was 6.3. The receptor had high specificity for prostaglandin F2alpha and had much lower affinities for other prostaglandins. Luteinizing and follicle-stimulating hormones had no effect on the prostaglandin F2alpha-receptor interaction.     

Beta nerve growth factor binding to PC12 cells. Association kinetics and cooperative interactions

The association kinetics of 125I beta nerve growth factor (NGF) binding to the PC12 clonal cell line have been examined in detail at 0.5 and 37 degrees C. These data were examined by utilizing a reversible second-order integrated rate equation, and the results were not consistent with a simple bimolecular process. Two association rates were required to explain the results adequately. At 37 degrees C, the faster component was estimated to have a second-order association rate constant of 1.4 X 10(7) M-1 s-1, while the rate constant for the slower component (3.8 X 10(6) M-1 s-1) was about 4-fold lower. As shown by others, the temperature dependence of the dissociation kinetics indicated that while the rapidly dissociating component was only slightly slowed by lowering the chase temperature to 0.5 degrees C, the second component was slowed by about 270-fold, from 8 X 10(-4) s-1 to 3 X 10(-6) s-1. The binding data that describe the slowly dissociating component were obtained by utilizing this differential temperature dependence and revealed a concave downward Scatchard plot. The binding parameters determined from computer analysis using a nonlinear fitting program (LIGAND) suggest that this component consists of (a) an interacting class of about 4000 sites/cell that have a first stoichiometric steady-state dissociation constant of 65 pM and a second stoichiometric interaction constant of 16 pM, indicative of positively cooperative interactions, and (b) a class of sites consistent with a ratio of sites/Kd of about 11.1 sites/(cell X pM). The steady-state binding results at 37 degrees C indicated only one class of binding sites (155,000 +/- 18,000 sites/cell) that had an apparent Kd of 0.52 +/- 0.03 nM. One class of sites was also observed at 0.5 degrees C, and the receptor concentration was found to be reduced (99,000 +/- 7600 sites/cell) while the Kd was increased (1.7 +/- 0.14 nM). A significant level of positively cooperative interactions was observed frequently at 37 degrees C that was not due to a failure to reach steady-state conditions, internalization, or degradation. Since cooperativity of binding was never observed at 0.5 degrees C, a membrane event may be involved. Determination of the contribution of the different classes of NGF receptors found on PC12 cells to the biological actions of NGF requires a clear understanding of their kinetic properties and their relationship to each other. The studies presented here indicate that their interactions are more complex than previously described.     

Thermodynamic and kinetic studies on the mechanism of binding of methylumbelliferyl glycosides to jacalin.

The binding of Artocarpus integrifolia lectin (jacalin) to 4-methylumbelliferyl (Meumb)-glycosides, Gal alpha Meumb, Gal beta Meumb, GalNAc alpha Meumb, GalNAc beta-Meumb, and Gal beta 3GalNAc beta Meumb was examined by extrinsic fluorescence quenching titration and stopped flow spectrofluorimetry. The binding was characterized by 100% quenching of fluorescence of Meumb-glycosides. Their association constants range from 2.0 x 10(4) to 1.58 x 10(6) M-1 at 15 degrees C. Entropic contribution is the major stabilizing force for avid binding of Meumb-glycosides indicating the existence of a hydrophobic site that is complementary to their methylumbelliferyl group. The second order association rate constants for interaction of these sugars with lectin at 15 degrees C vary from 8.8 x 10(5) to 3.24 x 10(6) M-1 S-1, at pH 7.2. The first order dissociation rate constants range from 2.30 to 43.0 S-1 at 15 degrees C. Despite the differences in their association rate constants, the overall values of association constants for these saccharides are determined by their dissociation rate constants. The second order rate constant for the association of Meumb-glycosides follows a pattern consistent with the magnitude of the activation energies involved therin. Activation parameters for association of all ligands illustrate that the origin of the barrier between binding of jacalin to Meumb-glycosides is entropic, and the enthalpic contribution is small. A correlation between these parameters and the structure of the ligands on the association rates underscores the importance of steric factors in determining protein saccharide recognitions.     

Properties of passive binding of calcium to endoplasmic reticulum from adipocytes.

Calcium binding to isolated adipocyte microsomes enriched in endoplasmic reticulum has been characterized. Binding was concentration-dependent, saturable, and totally dissociable. Steady state was reached within 20 min at all calcium concentrations tested. Three apparent classes of binding sites were identified in kinetic and steady state studies using calcium concentrations from 1 muM to 10 mM. The affinity constants (and maximum binding capacities) as determined by computer analysis for the three classes were 2.1 X 10(5) M-1 (0.28 nmol of calcium/mg of protein), 1.3 X 10(4) M-1 (1.1 nmol/mg), and 1.3 X 10(2) M-1 (35 nmol/mg). The dissociation rate constants for the high and intermediate affinity classes of sites were 1.6 X 10(-3) S-1, respectively, and the association rate constant for the high affinity sites was 8 X 10(2) M-1 S-1. The affinity constant calculated from the rate constants was 5.0 X 10(5) M-1 for the high affinity sites in agreement with the value obtained in studies at steady state. The three classes of binding sites were specific for calcium. Magnesium was a noncompetitive inhibitor of calcium binding to all three classes of sites with a Ki of 9 to 12 mM. Calcium binding at 1 muM calcium was 50% inhibited by 18 muM La3+, 600 muM Sr2+, or 2.7 mM Ba2+. These data represent the first analysis of passive calcium binding to endoplasmic reticulum from nonmuscular cells and the first report of corresponding rate constants for either endoplasmic or sarcoplasmic reticulum. The characteristics of the binding are consistent with the properties of calcium transport by endoplasmic reticulum of adipocytes. The characteristics and specificity of the calcium binding constitute further evidence that endoplasmic reticulum plays an important role in cellular calcium homeostasis.     

Binding of histidinal to histidinol dehydrogenase

One molecule of the enzymatic intermediate histidinal is firmly bound per subunit of histidinol dehydrogenase (EC 1.1.1.23) and protected against decomposition. The dissociation rate constant of the histidinal--histidinol dehydrogenase complex is estimated as 2.5 X 10(-5) S-1. Steady-state kinetic measurements studying the oxidation of histidinal to histidine and the reduction of histidinal to histidinol allow to calculate the association rate constants for histidinal. For both reactions the association rate constant is found as 1.9 X 10(6) M-1 S-1. Thus the dissociation constant of the histidinal--histidinol dehydrogenase complex is estimated to be of the order of 1.4 X 10(-11) M.     

Characteristics of Partially Purified Nerve Growth Factor Receptor

Receptors for the nerve growth factor protein (NGF) have been isolated from three cell types [embryonic chicken sensory neurons (dorsal root sensory ganglia; DRG), rat pheochromocytoma (PC12) and human neuroblastoma (LAN-1) cells] and have been shown to be similar with respect to equilibrium dissociation constants. The present results demonstrate that there are multiple molecular weight species for NGF receptors from DRG neurons and PC12 cells. NGF receptors can be isolated from DRG as four different molecular species of 228, 187, 125, and 112 kilodaltons, and PC12 cells as three molecular species of 203, 118, and 107 kilodaltons. The NGF receptors isolated from DRG show different pH-binding profiles for high- and low-affinity binding. High-affinity binding displays a bell-shaped pH profile with maximum binding between pH 7.0 and 7.9, whereas low-affinity binding is constant between pH 5.0 and 9.1, with a twofold greater binding at pH 3.6. At 22 degrees C, the association rate constant was found to be 9.5 +/- 1.0 X 10(6) M-1 s-1. Two dissociation rate constants were observed. The fast dissociating receptor has a dissociation rate constant of 3.0 +/- 1.5 X 10(-2) s-1, whereas the slow dissociating receptor constant was 2.4 +/- 1.0 X 10(-4) s-1. The equilibrium dissociation constants calculated from the ratio of dissociation to association rate constants are 2.5 X 109-11) M for the high-affinity receptor (type I) and 3.2 X 10(-9) M for the low-affinity receptor (type II). These values are the same as those determined by equilibrium experiments on the isolated receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equilibrium and kinetic studies of oxygen binding to the haemocyanin from the freshwater snail Lymnaea stagnalis.

The binding of oxygen by the haemocyanin of the gastropod Lymnaea stagnalis was studied by equilibrium and kinetic methods. The studies were performed under conditions in which the haemocyanin molecule was in the native state. Over the pH range 6.8-7.6, in the presence of 10mM-CaCl2 the haemocyanin bound O2 cooperatively. Over this pH range the haemocyanin molecule displayed a normal Bohr effect whereby the O2 affinity of the molecule decreased with a fall in the pH of the solution. The maximum slope of the Hill plot (hmax.) was 3.5, obtained at pH 7.5. An increase in the CaCl2 concentration from 5 to 20 mM at pH 6.8 resulted in a slight increase in the oxygen affinity, with hmax. remaining virtually unchanged. At constant pH and CaCl2 concentration, an increase in NaCl concentration from 0 to 50 mM resulted in a small decrease in O2 affinity, but a significant increase in the value of hmax. from 3.5 to 8.6. Temperature-jump relaxation experiments over a range of O2 concentrations produced single relaxation times. The dependence of the relaxation time on the reactant concentrations indicated a simple bimolecular binding process. The calculated association and dissociation rate constants for this process at pH 7.5 are 29.5 X 10(6) M-1 X S-1 and 49 S-1 respectively. The association rate constant kon was found to be essentially independent of pH and CaCl2 concentration. The dissociation rate constant, koff, however, increased with a decrease in the pH, but was also independent of CaCl2 concentration. These results indicate that the stability of the haemocyanin-O2 complex is determined by the dissociation rate constant.     

Intrinsic fluorescence changes and rapid kinetics of the reaction of thrombin with hirudin.

Stopped-flow fluorescence spectroscopy has been used to study the reaction of human alpha-thrombin with recombinant hirudin variant 1 (rhir) at 37 degrees C and an ionic strength of 0.125 M. A 35% enhancement in intrinsic fluorescence accompanied formation of the thrombin-rhir complex. Over one third of this enhancement corresponded to a structural change that could be induced by binding of either the NH2-terminal fragment (residues 1-51) or the COOH-terminal fragment (residues 52-65) of rhir. Three kinetic steps were detected for reaction of thrombin with rhir. At high rhir concentrations (greater than or equal to 3 microM), two intramolecular steps with observed rate constants of 296 +/- 5 s-1 and 50 +/- 1 s-1 were observed. By using the COOH-terminal fragment of rhir as a competitive inhibitor, it was possible to obtain an estimate of 2.9 x 10(8) M-1 s-1 for the effective association rate constant at low rhir concentrations. At higher ionic strengths, this rate constant was lower, which is consistent with the formation of the initial complex involving an ionic interaction. The mechanism for the reaction of both the COOH- and NH2-terminal fragments of rhir appeared to involve two steps. When thrombin was reacted with the COOH-terminal fragment at high concentrations (greater than or equal to 6 microM), the bimolecular step occurred within the dead time of the spectrometer and only one intramolecular step, with a rate constant of 308 +/- 5 s-1 was observed. At concentrations of NH2-terminal fragment below 50 microM, its binding to thrombin appeared to be a bimolecular reaction with an association rate constant of 8.3 x 10(5) M-1 s-1. In the presence of saturating concentrations of the COOH-terminal fragment, a 1.7-fold increase in this rate constant was observed. At concentrations of NH2-terminal fragment greater than 50 microM, biphasic reaction traces were observed which suggests a two-step mechanism. By comparing the reaction amplitudes and dissociation constants observed with rhir and its COOH-terminal fragment, it was possible to obtain approximate estimates for the values of the rate constants of different steps in the formation of the rhir-thrombin complex.     

Kinetics of carbon monoxide binding to singly reduced human methemoglobin

The kinetics of reaction of singly reduced methemoglobin (HbFe3(3+)Fe2+) with carbon monoxide have been investigated by the pulse radiolysis method. The rate constant for carbon monoxide binding to this form of hemoglobin is 4.1 X 10(6) M-1 S-1 at 24 degrees in our solutions. This value compares with existing values for various forms of hemoglobin ranging from 4 X 10(6) to 6.5 X 10(6) M-1 S-1. Addition of inositol hexaphosphate to the solutions results in a lower rate constant for carbon monoxide binding amounting to 1.1 X 10(5) M-1 S-1.