Studies of human hemoglobin intermediates. The double mixing method for studying the reactions of the species Hb4(CO) and Hb4(CO)2

Using the double mixing method we have studied the reactions of the partially liganded species (Hb4, Hb4L1, Hb4L2, Hb4L3) of normal human hemoglobin with carbon monoxide. In the first mixing, oxygen is removed from the species Hb4(O2) chi (CO) gamma and at the second mixing the species Hb4(CO) gamma reacts with CO. At 90% saturation of oxyHb with CO the main intermediate species are Hb4(CO)3 and Hb4(CO)2, and at 10% saturation Hb4 and Hb4(CO). The four CO-combination rate constants determined are: l'1 = 1 X 10(5) M-1 S-1, l'2 = 7 X 10(5) M-1 S-1, l'3 = 2 X 10(5) M-1 S-1 and l'4 = 4.8 X 10(6) M-1 S-1. The results indicate that there is no monotonic increase in the successive CO-combination rate constants. It is difficult to explain these results on the basis of the two-state model (Monod et al., 1965) or the stereochemical model of Perutz (1970).     

Kinetics of reduction of Clostridium pasteurianum rubredoxin by laser photoreduced spinach ferredoxin:NADP+ reductase and free flavins. Electron transfer within a protein-protein complex

The kinetics of reduction of oxidized Clostridium pasteurianum rubredoxin (Rdox) by free flavin semiquinones generated by the laser flash photolysis technique and by spinach ferredoxin:NADP+ reductase (FNR) semiquinone (also produced by flavin semiquinone reduction) have been investigated under anaerobic conditions. 5-Deazariboflavin semiquinone (5-dRf) rapidly reduces oxidized rubredoxin (Rdox) (k = 3.0 X 10(8) M-1 S-1) and oxidized ferredoxin:NADP+ reductase (FNRox) to the semiquinone level (k = 5.5 X 10(8) M-1 S-1). Lumiflavin semiquinone reduces Rdox more slowly (k = 1.3 X 10(7) M-1 S-1) and is not measurably reactive with FNRox. Absorption difference spectroscopy and difference CD indicate that Rdox and FNRox form a 1:1 complex at low ionic strength (10 mM), which is completely dissociated at higher ionic strength (310 mM). Apparent second order rate constants for reduction of Rdox in its free and complexed state by lumiflavin semiquinone are the same. Reduction of Rdox (both free and complexed) by free FNR semiquinone and intracomplex electron transfer were investigated using 5-dRf as the reductant. At I = 10 mM, a first order rate constant of 2.0 X 10(3) S-1 was obtained, which corresponds to the processes involved in intracomplex electron transfer from FNR semiquinone to Rdox. A second order reaction between free FNR semiquinone and complexed Rdox was also observed to occur (k = 5 X 10(7) M-1 S-1). At I = 310 mM, these reactions are not observed and the reaction of FNR semiquinone with free Rdox is second order (k = 4 X 10(6) M-1 S-1).     

Kinetics and mechanism of the reduction of horse heart ferricytochrome c by glutathione.

A detailed investigation of the reduction of cytochrome c by glutathione has shown that the reaction proceeds through several steps. A rapid combination of the reducing agent with the cytochrome leads to the formation of a glutathione-cytochrome intermediate in which the glutathione most likely interacts with the edge of the heme moiety. The electron transfer takes place in a subsequent slower step. Since cytochrome c(III) exists in two conformational forms at neutral pH [Kujundzic, N., & Everse, J. (1978) Biochem. Biophys. Res. Commun. 82, 1211], the reduction of cytochrome c by glutathione may be represented by cyt c(III) + GS- reversible K1 cyt c(III) ... GS- reversible k1 products cyt c*(III) + GS- reversible K2 cyt c*(III) ... GS- reversible k2 products At 25 degrees C, pH 7.5, and an ionic strength of 1.0 (NaCl), k1 = 1.2 X 10(-3) S-1, k2 = 2.0 X 10(-3) S-1, k1 = 2.9 X 10(3) M-1, and K2 = 5.3 X 10(3) M-1. The reaction is catalyzed by trisulfides, and second-order rate constants of 4.55 X 10(3) and 7.14 X 10(3) M-1 S-1 were obtained for methyl trisulfide and cysteine trisulfide, respectively.     

Distinct steps in the specific binding of tRNA to aminoacyl-tRNA synthetase. Temperature-jump studies on the serine-specific system from yeast and the tyrosine-specific system from Escherichia coli.

The kinetics of the interaction of tRNASer and seryl-tRNA synthetase from yeast as well as of tRNATyr and tyrosyl-tRNA synthetase from Escherichia coli have been investigated by temperature-jump experiments. It could be shown that complex formation proceeds in two distinct steps. This was demonstrated for both the first and the second binding site. The two-step mechanism was deduced from the characteristic concentration dependence of the relaxation times. Seryl-tRNA synthetase recombines with the first tRNA to form an intermediate complex (kI12, kI21), which is transformed in a fast reaction to the final 1:1 complex (kI23, kI32). At pH 7.2 with 0.1 M KCl the rate constants are: kI12 = 2.7 X 10(8) M-1 S-1; kI23, kI32). At pH 7.2 with 0.1 M KCl the rate constants are: kI12 = 2.7 x 10(8) M-1 S-1; kI21 = 220 S-1; kI23 = 760 S-1; kI32 = 330 S-1. The 1:1 complex can bind a second tRNA. At pH 7.2 without added salt the rate constants are: KII2 = 0.9 X 10(8) M-1 S-1; kII21 = 270 S-1; kII23 = 120 S-1; kII32 = 1250 S-1. The tyrosine-specific system behaves very similarly to the serine-specific system. Data are given for pH 7.2 (pH 6.0) for the binding of the second tRNA: kII12 = 1 X 10(8) (2.5 X 10(8)) M-1 S-1; kII21 = 470 (170) S-1; kII23 = 150 (530) S-1; kII32 = 1540 (720) S-1. The kinetic results are discussed in terms of their relevance to the recognition process and their relation to the anticooperative binding behaviour of tRNA to synthetase.     

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.     

Involvement of heparin chain length in the heparin-catalyzed inhibition of thrombin by antithrombin III

The mechanism of the heparin-promoted reaction of thrombin with antithrombin III was investigated by using covalent complexes of antithrombin III with either high-affinity heparin (Mr = 15,000) or heparin fragments having an average of 16 and 12 monosaccharide units (Mr = 4,300 and 3,200). The complexes inhibit thrombin in the manner of active site-directed, irreversible inhibitors: (Formula: see text) That is, the inhibition rate of the enzyme is saturable with respect to concentration of complexes. The values determined for Ki = (k-1 + k2)/k1 are 7 nM, 100 nM, and 6 microM when the Mr of the heparin moieties are 15,000, 4,300, 3,200, respectively, whereas k2 (2 S-1) is independent of the heparin chain length. The bimolecular rate constant k2/Ki for intact heparin is 3 X 10(8) M-1 S-1 and the corresponding second order rate constant k1 is 6.7 X 10(8) M-1 S-1, a value greater than that expected for a diffusion-controlled bimolecular reaction. The bimolecular rate constants for the complexes with heparin of Mr = 4,300 and 3,200 are, respectively, 2 X 10(7) M-1 S-1 and 3 X 10(5) M-1 S-1. Active site-blocked thrombin is an antagonist of covalent antithrombin III-heparin complexes: the effect is monophasic and half-maximum at 4 nM of antagonist against the complex with intact heparin, whereas the effect is weaker against complexes with heparin fragments and not monophasic. We conclude that virtually all of the activity of high affinity, high molecular weight heparin depends on binding both thrombin and antithrombin III to heparin, and that the exceptionally high activity of heparin results in part from the capacity of thrombin bound nonspecifically to heparin to diffuse in the dimension of the heparin chain towards bound antithrombin III. Increasing the chain length of heparin results in an increased reaction rate because of a higher probability of interaction between thrombin and heparin in solution.     

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.     

Steroid-protein interactions. Stopped flow fluorescence studies of the interaction between steroid hormones and progesterone-binding globulin.

Stopped flow fluorometry, measuring changes in the intrinsic fluorescence of progesterone-binding globulin (PBG), was used to determine the association and dissociation rates of the interaction of PBG with seven delta4-3-ketosteroids. The rates of formation and dissociation of the PBG-progesterone complex were measured as a function of concentration and temperature. At 20 degrees, kon = 8.7 X 10(7) M-1 S-1 and koff = 0.060 S-1. The association rate constants for progesterone, deoxycorticosterone, testosterone, testosterone acetate, and medrogestone were found to be the same within experimental error. The different affinities of PBG for these steroids result from the dissociation rate constants of the steroids which ranged from 0.43 S-1 for testosterone to 0.024 S-1 for medrogestone. Two corticosteroids, corticosterone and cortisol, were both bound somewhat more slowly (approximately 5 X 10(7) M-1 S-1). Reflecting their very low affinity for PBG both steroids dissociate very rapidly: corticosterone at 1.4 S-1 and cortisol at 90 S-1. The ratio of association to dissociation rate constants gave affinity constants in agreement with independently determined constants.     

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.     

Re-evaluation of the kinetics of lactate dehydrogenase-catalyzed chain oxidation of nicotinamide adenine dinucleotide by superoxide radicals in the presence of ethylenediaminetetraacetate.

The chain oxidation of lactate dehydrogenase-bound NADH initiated by superoxide radicals and propagated by oxygen was studied with pulse radiolysis. The kinetic parameters were re-evaluated in a system with carefully purified reagents (water and other chemicals) and in the presence of EDTA. The rate constant for the oxidation of the enzyme-bound NADH by O2- is calculated from the observed pseudo-first order disappearance of NADH and the chain length (molecules of NADH oxidized per O2- anion generated in the pulse). It is (1.0 +/- 0.2) X 10(5) M-1 S-1, consistent within a 13-fold variation in lactate dehydrogenase. NADH complex concentration and with varying chain length up to 6.1. Based on experiments with varying pH values from 4.5 to 9.0, the rate constant for oxidation of enzyme-bound NADH by HO2 is estimated to be 2.0 X 10(6) M-1 S-1.     

Mechanism of the interaction between ribosomal protein S1 and oligonucleotides.

The interaction of the ribosomal protein S1 from E. coli MRE 600 with oligonucleotides was studied by hydrodynamic, spectrophotometric, and kinetic methods. UV-difference spectra which are induced by the complex formation could be separated into a hyperchromic contribution originating from the nucleic acid moiety and a hypochromic contribution from the protein. Systematic determination of binding and rate constants was carried out by the temperature-jump relaxation technique. From the quantitative evaluation of the relaxation times and the relaxation amplitudes, the following conclusions could be drawn: The stoichiometry of the complex formation is one mole S1 per one mole oligonucleotide. The binding constant K, the recombination rate constant kR, and the dissociation rate constant kD, respectively, were measured at different temperatures. The values at 10 degrees C are K = 2 x 10(6) M-1, kR = 1.3 x 10(8) M-1S-1, kD = 65 s-1 for A(pA) 12 and K = 7.5 x 10(5) M-1, kR = 6.8 x 10(7) M-1S-1, kD = 90 S-1 for U(pU) 12. Discrepancies with data reported elsewhere are discussed. The stacking-unstacking equilibrium of the free oligonucleotide is frozen if the oligonucleotide is bound to the protein. The conformational change of the oligonucleotide does not occur in the form of a preequilibrium, but is induced after the primary binding step.     

Binding of simple carbohydrates and some of their chromophoric derivatives to soybean agglutinin as followed by titrimetric procedures and stopped flow kinetics

The number of carbohydrate-binding sites of the GalNAc-specific lectin is four per tetramer. The binding parameters of N-acetyl-D-galactosamine and methyl-N-acetyl-alpha-D- galactosaminide , were determined by titrating the perturbation in the absorption spectrum of the protein. For D-galactosides, it was necessary to use p-nitrophenyl-N-acetyl-beta-D- galactosaminide as an indicator in substitution titrations. The association constants K were determined at several temperatures yielding 2.4 X 10(4) M-1 at 25 degrees C with delta H degree' = -45 kJ mol-1 and delta S degree' = -67 J X K-1 mol-1 for methyl-N-acetyl-alpha-D- galactosaminide and 1.0 X 10(3) M-1 at 25 degrees C, delta H degree' = -38 kJ mol-1 and delta S degree' = -69 J X K-1 mol-1 for methyl-alpha-D-galactoside. The increase in K by a factor of 25 caused by the acetamido group is largely enthalpic . Whenever different methods were used to determine the association constant of a given compound, the agreement was excellent. The observed changes in absorption or fluorescence of all chromophoric carbohydrate derivatives used are specific for the binding of carbohydrates. For large aromatic beta- aglycons such as p-nitrophenyl or 4-methylumbelliferyl groups, the increase in K of the N-acetyl-D- galactosaminide moiety is by a factor of 2 or less, but for a large N-5-dimethylaminonaphthalene-1-sulfonyl (dansyl) group this factor is about 20 as compared with the acetyl group. The concomitant 10-fold increase in dansyl fluorescence, also observed with four other GalNAc-binding lectins together with a favorable and large delta S degree' = +60 J X K-1 mol-1 strongly point at the presence of a hydrophobic region in the vicinity of the carbohydrate-binding site. The results of stopped flow kinetics with 4-methylumbelliferyl-N-acetyl-beta-D- galactosaminide and the lectin are consistent with a simple mechanism for which k+ = 1.1 X 10(4) M-1 S-1 and k- = 0.4 S-1 at 25 degrees C. This k- is slower than for any monosaccharide-lectin complex reported so far.     

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.     

Cooperativity in the dissociation of nitric oxide from hemoglobin.

The dissociation of nitric oxide from hemoglobin, from isolated subunits of hemoglobin, and from myoglobin has been studied using dithionite to remove free nitric oxide. The reduction of nitric oxide by dithionite has a rate of 1.4 X 10(3) M-1 S-1 at 20 degrees in 0.05 M phosphate, pH 7.0, which is small compared with the rate of recombination of hemoglobin with nitric oxide (25 X 10(6) M-1 S-1 (Cassoly, R., and Gibson, Q. H. (1975) J. Mol. Biol. 91, 301-313). The rate of NO combination with chains and myoglobin was found to be 24 X 10(6) M-1 S-1 and 17 X 10(6) M-1 S-1, respectively. Hence, the observed progress curve of the dissociation of nitric oxide is dependent upon the dithionite concentration and the total heme concentration. Addition of excess carbon monoxide to the dissociation mixture reduces the free heme yielding a single exponential process for chains and for myoglobin which is dithionite and heme concentration independent over a wide range of concentrations. The rates of dissociation of nitric oxide from alpha chains, from beta chains, and from myoglobin are 4.6 X 10(-5) S-1, 2.2 X 10(-5) S-1, and 1.2 X 10(4) S-1, respectively, both in the presence and in the absence of carbon monoxide at 20 degrees in 0.05 M phosphate, pH 7.0. Analogous heme and dithionite concentration dependence is found for the dissociation of nitric oxide from tetrameric hemoglobin. The reaction is cooperative, the intrinsic rate constants for the dissociation of the 1st and 4th molecules of NO differing about 100-fold. With hemoglobin, replacement of NO by CO at neutral pH is biphasic in phosphate buffers. The rate of the slow phase is 1 X 10(-5) S-1 and is independent of pH. The amplitude of the fast phase increases with lowering of pH. By analogy with the treatment of the HbCO + NO reaction given by Salhany et al. (Salhany, J.M., Ogawa, S., and Shulman, R.G. (1975) Biochemistry 14, 2180-2190), the fast phase is attributed to the dissociation of NO from T state molecules and the slow phase to dissociation from R state molecules. Analysis of the data gives a pH-independent value of 0.01 for the allosteric constant c (c = Kr/Kt where Kr and Kt are the dissociation constants for NO from the R and T states, respectively) and pH-dependent values of L (2.5 X 10(7) at pH 7 in 0.05 M phosphate buffer). The value of c is considerably greater than that for O2 and CO. Studies of the difference spectrum induced in the Soret region by inositol hexaphosphate are also reported. This spectrum does not arise directly from the change of conformation between R and T states. The results show that if the equilibrium binding curve for NO could be determined experimentally, it would show cooperativity with Hill's n at 50% saturation of about 1.6.     

Reactions of nitrogen dioxide in aqueous model systems: oxidation of tyrosine units in peptides and proteins

By application of pulse radiolysis it was demonstrated that nitrogen dioxide (NO2.) oxidizes Gly-Tyr in aqueous solution with a strongly pH-dependent rate constant (k6 = 3.2 X 10(5) M-1 S-1 at pH 7.5 and k6 = 2.0 X 10(7) M-1 S-1 at pH 11.3), primarily generating phenoxyl radicals. The phenoxyl can react further with NO2. (k7 approximately 3 X 10(9) M-1 S-1) to form nitrotyrosine, which is the predominant final product in neutral solution and at low tyrosyl concentrations under gamma-radiolysis conditions. Tyrosine nitration is less efficient in acidic solution, due to the natural disproportionation of NO2., and in alkaline solutions and at high tyrosyl concentrations due to enhanced tyrosyl dimerization. Selective tyrosine nitration by interaction of NO2. with proteins (at pH 7 to 9) was demonstrated in the case of histone, lysozyme, ribonuclease A, and subtilisin Carlsberg. Nitrotyrosine developed slowly also under incubation of Gly-Tyr with nitrite at pH 4 to 5, where NO2. is formed by acid decomposition of HONO. It is recalled in this context that NO2.-induced oxidations, by regenerating NO2-, can propagate NO2./NO2- redox cycling under acidic conditions. Even faster than with tyrosine is the NO2.-induced oxidation of cysteine-thiolate (k9 = 2.4 X 10(8) M-1 S-1 at pH 9.2), involving the transient formation of cystinyl radical anions. The interaction of NO2. with Gly-Trp was comparably slow (k approximately 10(6) M-1 S-1), and no reaction was detectable by pulse radiolysis with Met-Gly and (Cys-Gly)2, or with DNA. Slow reactions of NO2. were observed with arachidonic acid (k approximately 10(6) M-1 S-1 at pH 9.0) and with linoleate (k approximately 2 X 10(5) M-1 S-1 at pH 9.4), indicating that NO2. is capable of initiating lipid peroxidation even in an aqueous environment. NO2.-Induced tyrosine nitration, using 50 microM Gly-Tyr at pH 8.2, was hardly inhibited, however, in the presence of 1 mM linoleate, and was not affected at all in the presence of 5 mM dimethylamine (a nitrosamine precursor). It is concluded that protein modifications, and particularly phenol and thiol oxidation, may be an important mechanism, as well as initiation of lipid peroxidation, of action of NO2. in biological systems.     

The electron-transfer reaction between azurin and the cytochrome c oxidase from Pseudomonas aeruginosa.

A stopped-flow investigation of the electron-transfer reaction between oxidized azurin and reduced Pseudomonas aeruginosa cytochrome c-551 oxidase and between reduced azurin and oxidized Ps. aeruginosa cytochrome c-551 oxidase was performed. Electrons leave and enter the oxidase molecule via its haem c component, with the oxidation and reduction of the haem d1 occurring by internal electron transfer. The reaction mechanism in both directions is complex. In the direction of oxidase oxidation, two phases assigned on the basis of difference spectra to haem c proceed with rate constants of 3.2 X 10(5)M-1-S-1 and 2.0 X 10(4)M-1-S-1, whereas the haem d1 oxidation occurs at 0.35 +/- 0.1S-1. Addition of CO to the reduced enzyme profoundly modifies the rate of haem c oxidation, with the faster process tending towards a rate limit of 200S-1. Reduction of the oxidase was similarly complex, with a fast haem c phase tending to a rate limit of 120S-1, and a slower phase with a second-order rate of 1.5 X 10(4)M-1-S-1; the internal transfer rate in this direction was o.25 +/- 0.1S-1. These results have been applied to a kinetic model originally developed from temperature-jump studies.     

Molecular mechanisms of olfactory reception. VI. Kinetic characteristics of camphor interaction with binding sites of rat olfactory epithelium

Camphor binding to a possible receptor of rat olfactory epithelium has been studied within the ligand concentration range 10(-11)-10(-6) M. At these concentrations camphor is bound by a set of receptors. They are distinguished by both the affinity to the ligand (K1 = 5 X 10(-10) M, K2 = 3.5 X 10(-8) M, K3 approximately equal to 10(-6) M) and their amount in the epithelium. The differences in the affinities are due to different values of the association rate constant of camphor (k1), which varies from 10(6) M-1 X s-1 for the receptors with high affinity up to 2 X 10(2) M-1 X s-1 for those with low affinity. These data are discussed in terms of equilibrium and kinetic models of the receptor-stimulus interaction.     

Kinetics of the inhibition of human leucocyte elastase by eglin from the leech Hirudo medicinalis.

The rate constants for the inhibition of human leucocyte elastase by eglin from the leech Hirudo medicinalis were determined by using a pre-steady-state kinetic approach. kon and koff for complex-formation and dissociation were 1 X 10(6)M-1 X S-1 and 8 X 10(-4)S-1 respectively. Ki was calculated as the ratio koff/kon = 8 X 10(-10)M, the binding of eglin to elastase was reversible and the inhibition mechanism was of the fully competitive type. The mechanistic properties of the system and the biological significance of the rate constants are discussed.     

Purification, characterization and ion binding properties of human brain S100b protein

Human brain S100b (beta beta) protein was purified using zinc-dependent affinity chromatography on phenyl-Sepharose. The calcium- and zinc-binding properties of the protein were studied by flow dialysis technique and the protein conformation both in the metal-free form and in the presence of Ca2+ or Zn2+ was investigated with ultraviolet spectroscopy, sulfhydryl reactivity and interaction with a hydrophobic fluorescence probe 6-(p-toluidino)naphthalene-2-sulfonic acid (TNS). Flow dialysis measurements of Ca2+ binding to human brain S100b (beta beta) protein revealed six Ca2+-binding sites which we assumed to represent three for each beta monomer, characterized by the macroscopic association constants K1 = 0.44 X 10(5) M-1; K2 = 0.1 X 10(5) M-1 and K3 = 0.08 X 10(5) M-1. In the presence of 120 mM KCl, the affinity of the protein for calcium is drastically reduced. Zinc-binding studies on human S100b protein showed that the protein bound two zinc ions per beta monomer, with macroscopic constants K1 = 4.47 X 10(7) M-1 and K2 = 0.1 X 10(7) M-1. Most of the Zn2+-induced conformational changes occurred after the binding of two zinc ions per mole of S100b protein. These results differ significantly from those for bovine protein and cast doubt on the conservation of the S100 structure during evolution. When calcium binding was studied in the presence of zinc, we noted an increase in the affinity of the protein for calcium, K1 = 4.4 X 10(5) M-1; K2 = 0.57 X 10(5) M-1; K3 = 0.023 X 10(5) M-1. These results indicated that the Ca2+- and Zn2+-binding sites on S100b protein are different and suggest that Zn2+ may regulate Ca2+ binding by increasing the affinity of the protein for calcium.     

Functional and subunit assembly properties of hemoglobin Alberta (alpha 2 beta 2(101) Glu----Gly)

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.