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.     

Reduction of laccase type 1 copper by 3,4-dihydroxyphenylalanine and other catechol derivatives

3,4-Dihydroxyphenylalanine (DOPA) is not a preferred substrate of Rhus vernicifera laccase, as rate constants for the anaerobic reduction of the type 1 cupric atom by L-DOPA (6.3 X 10(1) M-1 s-1), D-DOPA (2.6 X 10(1) M-1 s-1), and L-DOPA methyl ester (2.6 X 10(1) M-1 s-1) are considerably smaller than k1 (catechol) (7 X 10(2) M-1 s-1) and rate constants characteristic of numerous other nonphysiological organic substrates (25 degrees C, pH 7.0, I = 0.5 M). The reactions of DOPA derivatives with laccase are unique, however, in that a two-term rate law pertains: kobsd = k0 + k1[phenol]; k0(L-DOPA) = 7 X 10(-2) s-1. The reactivities of other catechol derivatives (pyrogallol, gallic acid, and methyl gallate) with laccase type 1 copper were also examined.     

Differential inhibitory properties of dog and human alpha 1-proteinase inhibitors

Dog alpha 1-proteinase inhibitor (alpha 1-PI) was found to be an effective inhibitor of bovine chymotrypsin and also of porcine pancreatic elastase as in the case of human inhibitor. The dog inhibitor inactivated both proteinases at a molar ratio of 1:1. However, compared to the human inhibitor, dog alpha 1-PI was a relatively poor inhibitor of bovine trypsin. The association rate constants (kass) of the interactions of dog alpha 1-PI with bovine chymotrypsin and with porcine elastase were determined to be 6.9 +/- 0.3 X 10(6) M-1 s-1 and 6.4 +/- 0.1 X 10(5) M-1 s-1, respectively. These values are 1.3- and 2.7-fold higher than the corresponding values for the human inhibitor. On the other hand, kass for the dog inhibitor with bovine trypsin (2.6 +/- 0.3 X 10(4)M-1 s-1) was found to be about 5 times smaller than that of the human inhibitor.     

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 3.4.22.2)-catalyzed hydrolysis of N-alpha-carbobenzoxyglycine p-nitrophenyl ester (ZGlyONp) have been determined between pH 3.0 and 9.5 (I = 0.1 M) at 21 +/- 0.5 degrees C. The results are consistent with the minimum three-step mechanism involving the acyl X enzyme intermediate E X P: (Formula: see text). The formation of the E X S complex may be regarded as a rapid pseudoequilibrium process; the minimum values for k+1 are 8.0 microM-1 s-1 (pH less than or equal to 3.5) and 0.40 microM-1 s-1 (pH greater than 6.0), and that for k-1 is 600 s-1 (pH independent). The pH profile of k+2/Ks (= kcat/Km; Ks = k-1/k+1) reflects the ionization of two groups with pK' values of 4.5 +/- 0.1 and 8.80 +/- 0.15 in the free enzyme. The pH dependence of k+2 and k+3 (measured only at pH values below neutrality) implicates one ionizing group in the acylation and deacylation step with pK' values of 5.80 +/- 0.15 and 3.10 +/- 0.15, respectively. As expected from the pH dependences of k+2/Ks (= kcat/Km) and k+2, the value of Ks changes with pH from 7.5 X 10(1) microM (pH less than or equal to 3.5) to 1.5 X 10(3) microM (pH greater than 6.0). Values of k-2 and k-3 are close to zero over the whole pH range explored (3.0 to 9.5). The pH dependence of kinetic parameters indicates that at acid pH values (less than or equal to 3.5), the k+2 step is rate limiting in catalysis, whereas for pH values higher than 3.5, k+3 becomes rate limiting. The observed ionizations probably reflect the acid-base equilibria of residues involved in the catalytic diad of papain, His159-Cys25. Comparison with catalytic properties of ficins and bromelains suggests that the results reported here may be of general significance for cysteine proteinase catalyzed reactions.     

Rate-determining folding and association reactions on the reconstitution pathway of porcine skeletal muscle lactic dehydrogenase after denaturation by guanidine hydrochloride

Reactivation of tetrameric porcine skeletal muscle lactic dehydrogenase after dissociation and extensive unfolding of the monomers by 6 M guanidine hydrochloride (Gdn . HCl) is characterized by sigmoidal kinetics, indicating a complex mechanism involving rate-limiting folding and association steps. For analysis of the association reactions, chemical cross-linking with glutaraldehyde may be used [Hermann, R., Jaenicke, R., & Rudolph, R. (1981) Biochemistry 20, 2195-2201]. The data clearly show that the formation of a dimeric intermediate is determined by a first-order folding reaction of the monomers with k1 = (8.0 +/- 0.1) x 10(-4) s-1. The rate constant of the association of dimers to tetramers which represents the second rate-limiting step on the pathway of reconstitution after guanidine denaturation, was then determined by reactivation and cross-linking experiments after dissociation in 0.1 M H3PO4 containing 1 M Na2SO4. The rate constant for the dimer association (which is the only rate-limiting step after acid dissociation) was k2 = (3.0 +/- 0.5) x 10(4) M-1 s-1. On the basis of the given two rate constants, the complete reassociation pattern of porcine lactic dehydrogenase after dissociation and denaturation in 6 M Gdn . HCl can be described by the kinetic model (formula: see text).     

Tissue-type plasminogen activator and urokinase: differences in the reaction pattern with the active-site titrant 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride

The applicability of 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride (MUGB) as active-site titrant for tissue-type plasminogen activator (t-PA) was studied in comparison to urokinase. Although t-PA was capable of cleaving MUGB, active-site titration of t-PA (one-chain form as well as two-chain form) with MUGB was not possible, whereas MUGB titration of urokinase could be performed. We therefore studied the kinetics of the interaction of these two plasminogen activators with MUGB. The equilibrium dissociation constant, KS, for the interaction between MUGB and urokinase was 2.9 X 10(-6) M, and for the interaction with t-PA 3.13 X 10(-5) M. However, one main requirement for active-site titration, namely a stable acyl enzyme intermediate (ES'), was only fulfilled for MUGB urokinase but not for MUGB t-PA. Whereas for the reaction of MUGB and urokinase the first-order acylation rate constant k2 was found to be about 10(6)-times higher than the first-order deacylation rate constant k3 (k2 = 3.76 X 10(-1) s-1, k3 = 3.7 X 10(-7) s-1), the k2/k3 ratio for the reaction of MUGB and t-PA (one- and two-chain form) was 0.77 to 3.85. Therefore, urokinase and t-PA differ in their reaction with this fluorogenic substrate and MUGB cannot be used for active-site titration of tPA.     

Studies on rat liver plasma membrane. Altered protein and phospholipid metabolism after injection of D-galactosamine.

The displacement of NADH from cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by using NAD+, 1,10-phenanthroline, ADP-ribose, deamino-NAD+ and pyridine-3-aldehyde-adenine dinucleotide as displacing agents, by following the decrease in fluorescence as a function of time. The data obtained could be fitted by assuming two first-order processes were occurring, a faster process with an apparent rate constant of 0.85 +/- 0.20 s-1 and a relative amplitude of 60 +/- 10% and a slower process with an apparent rate constant of 0.20 +/- 0.05 s-1 and a relative amplitude of 40 +/- 10% (except for pyridine-3-aldehyde-adenine dinucleotide, where the apparent rate constant for the slow process was 0.05 s-1). The displacement rates did not change significantly when the pH was varied from 6.0 to 9.0. Kinetic data are also reported for the dependence of the rate of binding of NADH to the enzyme on the total concentration of NADH. Detailed arguments are presented based on the isolation and purification procedures, the equilibrium coenzyme-binding studies and the kinetic data, which lead to the following model for the release of NADH from the enzyme: (formula: see article). The parameters that best fit the data are: k + 1 = 0.2 s-1; k - 1 = 0.05 s-1; k + 2 = 0.8 s-1 and k - 2 = 5 X 10(5)litre-mol-1-s-1. The slow phase of the NADH release is similar to the steady-state turnover number for substrates such as acetaldehyde and propionaldehyde and appears to contribute significantly to the limitation of the steady-state rate.     

Synthesis and characterization of a new fluorogenic active-site titrant of serine proteases

The molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-[N'-(4-carboxyphenyl)thioureido[spirop]isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue of p-nitrophenyl p-guanidino-benzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of trypsin in phosphate-buffered saline, pH 7.2. The rate of acylation with FDE is extremely fast (k2 = 1.05 s-1) and the rate of deacylation extremely slow (k3 = 1.66 X 10(-5) s-1). The Ks is 3.06 X 10(-6) M, and the Km(app) is 4.85 X 10(-11) M. With two of the serine proteases involved in fibrinolysis, the rate of acylation with FDE is also fast, K2 = 0.112 s-1 for urokinase and 0.799 s-1 for plasmin, and the rate of deacylation is slow, k3 = 3.64 X 10(-4) s-1 for urokinase and 6.27 X 10(-6) s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 X 10(-5) M, and the first-order rate constant for spontaneous hydrolysis is 5.1 X 10(-6) s-1. The major difference between FDE and NPGB is the detectability of the product in an active-site titration. p-Nitrophenol can be detected at concentrations no lower than 10(-6) M whereas fluorescein can be detected at concentrations as low as 10(-12) M. Thus, FDE should be useful in quantitatively assaying serine proteases as very low concentrations.     

Reactions of prostaglandin H synthase in the presence of the stabilizing agents diethyldithiocarbamate and glycerol

Both cyclooxygenase and peroxidase reactions of prostaglandin H synthase were studied in the presence and absence of diethyldithiocarbamate and glycerol at 4 degrees C in phosphate buffer (pH 8.0). Diethyldithiocarbamate reacts with the high oxidation state intermediates of prostaglandin H synthase; it protects the enzyme from bleaching and loss of activity by its ability to act as a reducing agent. For the reaction of diethyldithiocarbamate with compound I, the second-order rate constant k2,app, was found to fall within the range of 5.8 x 10(6) +/- 0.4 x 10(6) M-1.s-1 less than k2,app less than 1.8 x 10(7) +/- 0.1 x 10(7) M-1.s-1. The reaction of diethyldithiocarbamate with compound II showed saturation behavior suggesting enzyme-substrate complex formation, with kcat = 22 +/- 3 s-1, Km = 67 +/- 10 microM, and the second-order rate constant k3,app = 2.0 x 10(5) +/- 0.2 x 10(5) M-1.s-1. In the presence of both diethyldithiocarbamate and 30% glycerol, the parameters for compound II are kcat = 8.8 +/- 0.5 s-1, Km = 49 +/- 7 microM, and k3,app = 1.03 x 10(5) +/- 0.07 x 10(5) M-1.s-1. The spontaneous decay rate constants of compounds I and II (in the absence of diethyldithiocarbamate) are 83 +/- 5 and 0.52 +/- 0.05 s-1, respectively, in the absence of glycerol; in the presence of 30% glycerol they are 78 +/- 5 and 0.33 +/- 0.02 s-1, respectively. Neither cyclooxygenase activity nor the rate constant for compound I formation using 5-phenyl-4-pentenyl-1-hydroperoxide is altered by the presence of diethyldithiocarbamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of carboxymethylation of histidine hydantoin.

The reaction of the imidazole group of histidine hydantoin with bromoacetate was studied as a model for carboxymethylation of histidine residues in proteins. pK values of 6.4 and 9.1 (25 degrees C) and apparent heats of ionization of 7.8 and 8.7 kcal/mol were determined for the imidazole and hydantoin rings, respectively. At pH values corresponding to the isoelectric points for histidine hydantoin, the rates of carboxymethylation at 12, 25, 37, and 50 degrees C were determined; the modified hydantoins were hydrolyzed to the corresponding histidine derivatives for quantitative amino acid analysis. At pH 7.72 and 25 degrees C, the imidazole tele-N was alkylated (k = 3.9 X 10(-5) M-1 s-1) twice as fast as the pros-N. The monocarboxymethyl derivatives were carboxymethylated at the same rate at the pros-N (k = 2.1 X 10(-5) M-1 s-1) but 3 times faster at the tele-N (k = 11 X 10(-5) M-1 s-1). The enthalpies of activation determined for carboxymethylation of the imidazole ring and its monocarboxymethyl derivatives were similar (15.9 +/- 0.7 kcal/mol). delta S for the four carboxymethylations was -25 +/- 2 eu. The electrostatic component of delta S (delta S es) was calculated from the influence of the dielectric constant on the reaction rate at 25 degrees C. delta S es was slightly negative (-4 +/- 1 eu) for mono- or dicarboxymethylations, indicating some charge separation in the transition state. The nonelectrostatic entropy of activation was -21 +/- 2 eu for all four carboxymethylations.     

A kinetic study of cyclic adenosine 3':5'-monophosphate binding and mode of activation of protein kinase from Drosophila melanogaster embryos.

Cyclic AMP-dependent protein kinase and its regulatory subunit were isolated from Drosophila melanogaster embryos. The profiles of cyclic AMP binding by these proteins were significantly different. In order to explain such a difference and to find the mode of enzyme activation by cyclic AMP, a kinetic study of cyclic AMP binding was carried out. First, the association rate constant k1 and dissociation rate constant k-1 in the cyclic AMP-regulatory subunit interaction at 0 degrees C were estimated to be 2.3 X 10(6)M-1s-1 and 1.1 X 10(-3)s-1, respectively. Secondly, the three possible modes of enzyme activation by cyclic AMP were mathematically considered and could be described by a unique formula: r=APt + BQt (A + B=1) in which the parameters A, B, P, and Q are equivalent to rate constants in the sense that the rate constants are simply expressed by these parameters. Thirdly, the values of the parameters and subsequently the values of rate constants involved in the possible mechanisms were evaluated using a curve-fitting technique and compared with experimental observation. It was then found that the following mechanism was the only one which fitted the experimental observations. Namely, RC + L k3 equilibrium k-3 LRC k4 equilibrium k-4 RL + C where R, C, and L represent the regulatory and catalytic subunits and cyclic AMP as a ligand. Thus, our results indicate that in the presence of cyclic AMP the active enzyme (C) is released from a ternary intermediate which is the primary product of the cyclic AMP-holoenzyme interaction. The estimated values of the rate constants are: k3=3.5 X 10(6)M-1s-1;k-3=7.3 X 10(-1)s-1;and k4=3.8 X 10(-2)s. These estimates indicate that the reaction LRC leads to RL + C is relatively slow and limits the rate of the overall reaction. By comparing k-3 and k4, it is apparent that a large part of newly formed ternary intermediate reverts to the holoenzyme.     

Peptide models of protein metastable binding sites: competitive kinetics of isomerization and hydrolysis

alpha 2-Macroglobulin and the complement components C3 and C4 each contain a metastable binding site that is essential for covalent attachment. Two cyclic peptides are useful models of these unusual protein sites. Five-membered lactam 1 (CH3CO-Gly-Cys-Gly-Glu-Glp-Asn-NH2) contains an internal residue of pyroglutamic acid (Glp). Fifteen-membered thiolactone 2 (CH3CO-Gly-Cys-Gly-Glu-Glu-Asn-NH2 15-thiolactone) contains a thiol ester bond between Cys-2 and Glu-5. These isomeric hexapeptides are spontaneously interconverted in water. Competing with the two isomerization reactions are three reactions involving hydrolysis of 1 and 2. These five processes were found to occur simultaneously under physiologic conditions (phosphate-buffered saline, pH 7.3, 37 degrees C). Best estimates of the five rate constants for these apparent first-order reactions were obtained by comparing the observed molar percentages of peptides 1-4 with those calculated from a set of exponential equations. Both isomerization reactions (ring expansion of 1 to 2, k1 = 6.4 X 10(-5) s-1; ring contraction of 2 to 1, k-1 = 69 X 10(-5) s-1) proceeded faster than any of the hydrolysis reactions: alpha-cleavage of 1 with fragmentation to form dipeptide 3 (k2 = 3.3 X 10(-5) s-1), gamma-cleavage of 1 with ring opening to yield mercapto acid 4 (k3 = 0.35 X 10(-5) s-1), and hydrolysis of 2 with ring opening to give 4 (k4 = 1.9 X 10(-5) s-1). The isomerization rate ratio (k1/k-1 = 10.9) agreed with the isomer ratio at equilibrium (1:2 = 11 starting from 1 and 10 starting from 2). The alpha/gamma regioselectivity ratio (k2/k3 = 9.7) for hydrolysis of the internal Glp residue of 1 was consistent with results for model tripeptides. Part of the chemistry of the protein metastable binding sites can be explained by similar isomerization and hydrolysis reactions.     

Kinetics of slow, tight-binding inhibitors of angiotensin converting enzyme

Five phosphorus-containing inhibitors of angiotensin converting enzyme were found to exhibit slow, tight-binding kinetics by using furanacryloyl-L-phenylalanylglycylglycine as substrate at pH 7.50 and T = 25 degrees C. Two of the inhibitors, (O-ethylphospho)-Ala-Pro (2) and (O-isopropylphospho)-Ala-Pro (3), are found to follow at minimum a two-step mechanism of binding (mechanism B) to the enzyme. This mechanism consists of an initial fast formation of a weaker enzyme-inhibitor complex (Ki = 130 nM for 2 and 180 nM for 3) followed by a slow reversible isomerization to a tighter complex with measurable forward (K3) and reverse (k4) rate constants (k3 = 4.5 X 10(-2) s-1 for 2 and 5.4 X 10(-2) s-1 for 3; k4 = 9.2 X 10(-3) s-1 for 2 and 3.5 X 10(-3) s-1 for 3). For the remaining three inhibitors, phospho-Ala-Pro (1), (O-benzyl-phospho)-Ala-Pro (4), and (P-phenethylphosphono)-Ala-Pro (5), a one-step binding mechanism (mechanism A) is observed under the conditions of the experiment. The second-order rate constants k1 (M-1 s-1) for the binding of these inhibitors to converting enzyme are found to have values more than 3 orders of magnitude lower than the diffusion-controlled limit for a bimolecular reaction involving the enzyme, viz., 3.9 X 10(5) for 1, 2.2 X 10(5) for 4, and 4.8 X 10(5) for 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic studies on the interaction of eglin c with human leukocyte elastase and cathepsin G

We have investigated the inhibition of human leukocyte elastase and cathepsin G by recombinant Eglin c under near physiological conditions. The association rate constants k on of Eglin c for elastase and cathepsin G were 1.3 X 10(7) M-1 s-1 and 2 X 10(6) M-1 s-1, respectively. Under identical conditions, the k on for the association of human plasma alpha 1-proteinase inhibitor with the two leukocproteinases were 2.4 X 10(7) M-1 s-1 and 10(6) M-1 s-1, respectively. The consistency of these data could be verified using a set of competition experiments. The elastase-Eglin c interaction was studied in greater detail. The dissociation rate constant k off was determined by trapping of free elastase from an equilibrium mixture of elastase and Eglin c with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The rate of dissociation was very low (k off = 3.5 X 10(-5) s-1). The calculated equilibrium dissociation constant of the complex, Ki(calc) = k off/k on, was found to be 2.7 X 10(-12) M. Ki was also measured by adding elastase to mixtures of Eglin c and substrate and determining the steady-state rates of substrate hydrolysis. The Ki determined from these experiments (7.5 X 10(-11) M) was significantly higher than Ki(calc). This discrepancy might be explained by assuming that the interaction of Eglin c with elastase involves two steps: a fast binding reaction followed by a slow isomerization step. From the above kinetic constants it may be inferred that at a therapeutic concentration of 5 X 10(-7) M, Eglin c will inhibit leukocyte elastase in one second and will bind this enzyme in a "pseudo-irreversible" manner.     

Purification and characterization of a novel inhibitor of urokinase from human urine. Quantitation and preliminary characterization in plasma

Urokinase-related proteins in human urine occur mainly as a 1:1 complex of urokinase with an inhibitor (Stump, D. C., Thienpont, M., and Collen, D. (1986) J. Biol. Chem. 261, 1267-1273). BALB/c mice were immunized with this urokinase-urokinase inhibitor complex and spleen cells fused with mouse myeloma cells, resulting in hybridomas producing monoclonal antibodies. Three antibodies reacting with the complex but not with urokinase were utilized to develop a sensitive (0.5 ng/ml) enzyme-linked immunosorbent assay for the urokinase inhibitor, which was used for monitoring its purification by chromatography on zinc chelate-Sepharose, concanavalin A-Sepharose, SP-Sephadex C-50, and Sephadex G-100. A homogenous glycoprotein of apparent Mr 50,000 was obtained with a yield of 40 micrograms/liter urine and a purification factor of 320. One mg of the purified protein inhibited 35,000 IU of urokinase within 30 min at 37 degrees C. This protein was immunologically related to both the purified urokinase-urokinase inhibitor complex and to the inhibitor portion dissociated from it by nucleophilic dissociation. It was immunologically distinct from all known protease inhibitors, including the endothelial cell-derived fast-acting inhibitor of tissue-type plasminogen activator, the placental inhibitor of urokinase and protease nexin. In electrophoresis the protein migrated with beta-mobility. Inhibition of urokinase occurred with a second order rate constant (k) of 8 X 10(3) M-1 s-1 in the absence and of 9 X 10(4) M-1 s-1 in the presence of 50 IU of heparin/ml. The urokinase inhibitor was inactive towards single-chain urokinase-type plasminogen activator and plasmin, but it inhibited two-chain tissue-type plasminogen activator with a k below 10(3) M-1 s-1 and thrombin with a k of 4 X 10(4) M-1 s-1 in the absence and 2 X 10(5) M-1 s-1 in the presence of heparin. The concentration of this urokinase inhibitor in plasma from normal subjects determined by immunoassay was 2 +/- 0.7 micrograms/ml (mean +/- S.D., n = 25). The protein purified from plasma by immunoabsorption had the same Mr, amino acid composition, and immunoreactivity as the urinary protein. Furthermore, when urokinase was added to plasma, time-dependent urokinase-urokinase inhibitor complex formation was observed at a rate similar to that observed for the inhibition of urokinase by the purified inhibitor from urine. This urokinase inhibitor, purified from human urine, most probably represents a new plasma protease inhibitor.     

A kinetic study of protein-protein interactions.

Kinetic studies have been carried out of the monomer-dimer interaction of insulin, beta-lactoglobulin, and alpha-chymotrypsin using stopped-flow and temperature-jump techniques. The pH indicators bromothymol blue, bromophenol blue, and phenol red were used to monitor pH changes associated with the monomer-dimer interaction. In all three cases a kinetic process was observed which could be attributed to a simple monomer-dimer equilibrium, and association (k1) and dissociation (k-1) rate constants were determined. The results obtained are as follows: for insulin at 23 degrees C, pH 6.8, 0.125 M KNO3, k1 = 1.14 X 10(8) M-1 s-1, k-1 - 1.48 X 10(4)s(-1); for beta-lactoglobulin AB at 35 degrees C, pH 3.7, 0.025 M KNO3, d1 = 4.7 X 10(4) M-1 s-1, k-1 = 2.1 s-1; for alpha-chymotrypsin at 25 degreesC, pH 4.3, 0.05 M KNO3 k1 - 3.7 X 10(3) M-1 s-1, k-1 - 0.68 s-1. The kinetic behavior of the separated beta-lactoglobulin A and B was similar to that of the mixture. In the case of chymotrypsin, bromophenol blue was found to activate the enzyme catalyzed hydrolysis of p-nitrophenyl acetate, and a rate process was observed with the temperature jump which could be attributed to a conformational change of the indicator-protein complex. The association rate constant for dimer formation of insulin approaches the value expected for a diffusion-controlled process, while the values obtained for the other two proteins are below those expected for a diffusion-controlled reaction unless unusally large steric and electrostatic effects are present.     

Nitrogenase of Klebsiella pneumoniae. Kinetics of the dissociation of oxidized iron protein from molybdenum-iron protein: identification of the rate-limiting step for substrate reduction.

Stopped-flow spectrophotometry and e.p.r. spectroscopy were used to study the kinetics of reduction by dithionite of the oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox.) in the presence of MgADP at 23 degrees C at pH 7.4. The active reductant, SO2.-, produced by the predissociation of S2O4(2-) in equilibrium 2SO2.-, reacts with Kp2ox. (MgADP)2, with k4 = 3.0 X 10(6) +/- 0.4 X 10(6) M-1 X s-1. The inhibition of this reaction by the Mo-Fe protein (Kp1) has enabled the rate of dissociation of Kp2ox. (MgADP)2 from Kp1+ (the Kp2-binding site on Kp1) to be measured (k-3 = 6.4 +/- 0.8 s-1). Comparison with the steady-state rate of substrate reduction shows that the dissociation (k-3) of the complex Kp2ox. (MgADP)2-Kp1+, which is formed after MgATP-induced electron transfer from Kp2 to Kp1+, is the rate-limiting step in the catalytic cycle for substrate reduction.     

Intermolecular charge transfer involving tryptophan, tyrosine and three electron-bonded intermediates derived from methionine

Oxidation processes of radiation-generated three-electron-bonded intermediates derived from methionine Met2[S+...S] and Met[S...X] (X=Cl,Br) were investigated through reaction with tryptophan and tyrosine, using the optical pulse radiolysis method. Bimolecular rate constants have been measured for the reactions Met2[S+...S] with TrpH(k=3.8 x 10(8) dm3 mol-1 s-1 and k = 4.9 X 10(8) dm3 mol-1 s-1 at at ph 1 and 4.3, respectively) and Met2[S+...S] with tyrosine, k=3.8 x 10(7) dm3 mol-1 s-1. Rate constants for intermolecular transformation of Met[S...Br] and Met[S...Cl] into TrpH+. or Trp. were also estimated. They varied from 4.7 X 10(8) dm3 mol-1 s-1 (bromide species) to 1.0 X 10(9)dm3 mol-1 s-1 (chloride species). It has also been established azide radicals N-6, N.3 in contrast to dihalide radicals (X-2) do not form transients of Met[S...X] (X = N3)-type. However, oxidation of N-3 by Met2[S+...S] occurs with a bimolecular rate constant of 2.8 X 10(8) dm3 mol-1 s-1. These results are discussed in the light of some equilibria which have been proposed earlier for methionine-halide systems.     

The porcine ovarian luteinizing hormone/human chorionic gonadotropin receptor II. Is the purified receptor an oligomer of identical subunits?

Purified porcine luteinizing hormone/human chorionic gonadotropin receptors were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis following reduction and thermal denaturation and stained with Coomassie Brilliant Blue. A major protein of Mr = 77 +/- 4 X 10(3) and a minor protein of Mr = 66 +/- 4 X 10(3) were observed. Iodoreceptor proteins were resolved into a major component of Mr = 77 +/- 3 X 10(3) and a minor component of Mr = 62 +/- 5 X 10(3) after reduction and thermal denaturation. In the absence of reduction, the iodoreceptor had a major component of Mr 63 +/- 3 X 10(3). Purified human chorionic gonadotropin specifically transferred part of the iodoreceptor from the Mr = 63 X 10(3) species to an Mr = 110-120 X 10(3) species. Purified receptors were analyzed by nondenaturing polyacrylamide gel electrophoresis and identified by specific binding of iodo-human chorionic gonadotropin. Three binding species with approximate Mr = 60 X 10(3), 130 X 10(3), and 260 X 10(3) were identified. Iodoreceptors co-migrated with the Mr = 60 X 10(3) species under the same conditions. Similar results were obtained following renaturation of receptors separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without reduction and thermal denaturation. These results suggest for the first time that the porcine corpus luteum luteinizing hormone/human chorionic gonadotropin receptor may be a hormone binding monomer of Mr = 60-65 X 10(3), and that the monomer may associate to form hormone binding polymeric receptor complexes.     

Effect of monovalent cations on the pre-steady-state kinetic parameters of the plasma protease bovine activated protein C

Activated bovine plasma protein C (APC) was not reactive with the substrate p-nitrophenyl p-guanidinobenzoate (NPGB) in the absence of cations. In the presence of increasing concentrations of Na+, the acylation rate constant, k2,app, at 7 degrees C, progressively increased from 0.32 +/- 0.03 s-1 at 12.5 mM Na+ to 1.15 +/- 0.10 s-1 at 62.5 mM Na+. A linear dependence of the reciprocal of k2,app with [Na+]-2 was observed, indicating that at least two monovalent cation sites, or classes of sites, are necessary for the catalytic event to occur. From this latter plot, the k2,max for APC catalysis of NPGB hydrolysis, at saturating [Na+] and [NPGB], was calculated to be 1.21 +/- 0.10 s-1, and the Km for Na+ was found to be 21 +/- 3 mM. The dissociation constant, Ks, for NPGB to APC, at 7 degrees C, was not altered as [Na+] was increased, yielding a range of values of 18.5 X 10(-5) to 19.9 X 10(-5) M as [Na+] was varied from 12.5 to 62.5 mM. The deacylation rate constant, k3, for p-guanidinobenzoyl-APC hydrolysis was also independent of [Na+], with a value of (3.8 +/- 1.0) X 10(-3) s-1 in the absence of Na+ or in the presence of concentrations of Na+ up to 200 mM. Identical kinetic behavior was observed when Cs+ was substituted for Na+ in the above enzymic reaction. The pre-steady-state kinetic parameters were calculated according to the same methodology as described above.(ABSTRACT TRUNCATED AT 250 WORDS)