Search PubMed⌕ Search

Biomedical subjects

J S Olson

Publications and source records attributed to J S Olson.

At least 109 records · Page 6Linked to original sources
PubMed1984

The reaction of xanthine oxidase with lumazine. Characterization of the reductive half-reaction.

The optical and fluorescence characteristics of lumazine (2, 4-dihydroxypteridine) and violapterin (2,4,6-trihydroxypteridine) are described. The reduction of xanthine oxidase by excess lumazine is markedly biphasic. The initial rapid phase is about 100-fold more rapid than Vmax and reflects the reaction of the enzyme with the first equivalent of lumazine; it is accompanied by the appearance of long wavelength absorbance of the Mo(IV) violapterin charge transfer species together with partial reduction in absorbance at 450 nm, reflecting a partial transfer of electrons from the Mo to the flavin and iron-sulfur center. A loss of substrate fluorescence and a burst of fluorescence due to enzyme bound product also occur during this phase. The second phase is rate-controlled by the dissociation of violapterin from the reduced enzyme. It is accompanied by a variable loss of the charge-transfer band, further reduction of the flavin and iron-sulfur chromophores and of substrate fluorescence, and the appearance of fluorescence of unbound product. This phase correlates well with the steady state parameters. The variation in kinetic behavior with pH is qualitatively explained by a marked decrease in the affinity of the enzyme as the pH is raised; Vmax is almost unaffected by pH. The temporal sequence of kinetic events reveals at least three steps in the reaction of xanthine oxidase with the first equivalent of lumazine.

Anaerobiosis↗
PubMed1984

A quantitative description in three dimensions of oxygen uptake by human red blood cells.

Oxygen uptake by human erythrocytes has been examined both experimentally and theoretically in terms of the influence of unstirred solvent layers that are adjacent to the cell surface. A one-dimensional plane sheet model has been compared with more complex spherical and cylindrical coordinate schemes. Although simpler and faster, the plane sheet algorithm is an inadequate representation when unstirred solvent layers are considered. The cylindrical disk model most closely represents the physical geometry of human red cells and is required for a quantitative analysis. In our stopped-flow rapid mixing experiments, the thickness of the unstirred solvent layer expands with time as the residual turbulence decays. This phenomenon has been quantified using a formulation based on previously developed hydrodynamic theories. An initial 10(-4) cm unstirred layer is postulated to occur during mixing and expand rapidly with time by a (t)0.5 function when flow stops. This formula, in combination with the three-dimensional cylinder scheme, has been used to describe quantitatively uptake time courses at various oxygen concentrations, two different external solvent viscosities, and two different internal heme concentrations.

Diffusion↗
PubMed1983

Ligand binding to heme proteins. An evaluation of distal effects.

The O2, CO, and alkyl isocyanide-binding properties of a variety of vertebrate and invertebrate heme proteins have been compared in detail to those of protoheme mono-3-(1-imidazoyl)-propylamide monomethyl ester in aqueous suspensions of soap micelles. The proteins examined include: cytochrome P-450cam from Pseudomonas putida, beef heart cytochrome c oxidase, yeast cytochrome c peroxidase, alpha and beta subunits of human hemoglobin, sheep hemoglobin, carp hemoglobin, sperm whale myoglobin, horse heart myoglobin, a monomeric hemoglobin from Glycera dibranchiata, erythrocruorin from Chironomusthummii, soybean leghemoglobin, and several hemoglobins that lack distal histidines. The smallest bimolecular rates were observed for cytochrome P-450 containing bound camphor, cytochrome c oxidase, and cytochrome c peroxidase. In the case of P-450, the extremely low isonitrile binding rates (approximately 1 M-1 S-1 at 20 degrees C) are due to steric exclusion by bound camphor molecules. For the oxidase and peroxidase, inhibition of CO and isonitrile binding appears to be due to the polar nature of the active sites. In the cases of animal hemoglobins and myoglobins, the sixth coordination positions appear to be designed to accommodate diatomic molecules with no steric hindrance by distal protein residues. Protein resistance to the diffusion of CO and O2 does not limit the observed association rate constants. In contrast, ligands containing three or more atoms are sterically hindered both in their final bound positions and during diffusion to the active site. The magnitude of this hindrance (greater than or equal to 2 kcal/mol) exhibits a complex dependence on ligand size and shape. The most important protein residue appears to be His E7. In addition to restricting the size of the sixth coordination position, the distal histidine is also capable of forming a hydrogen bond with bound oxygen molecules. The strength of this hydrogen bond was estimated to be -2 and -1 kcal/mol for mammalian myoglobins and hemoglobins, respectively, and accounts for the smaller CO/O2 partition constants (M values) observed for these proteins in comparison to the constants observed for pentacoordinate model heme compounds.

Animals↗
PubMed1983

Rates of ligand binding to periplasmic proteins involved in bacterial transport and chemotaxis.

The ligand reactions of three binding proteins involved in bacterial transport and chemotaxis have been examined by stopped flow, rapid mixing techniques. The processes measured were: L-arabinose, D-galactose, and D-fucose binding to the Escherichia coli L-arabinose-binding protein; L-histidine binding to the Salmonella typhimurium L-histidine-binding protein; and D-maltose, maltotriose, cyclic maltohexaose, and cyclic maltoheptaose binding to the E. coli D-maltose-binding protein. Changes in tryptophan fluorescence were monitored, and the resultant time courses were analyzed quantitatively in terms of a simple one-step binding process. The fitted association rate constants for sugar binding are all about 1-3 X 10(7) M-1 s-1; variation in the affinity constants is expressed primarily by changes in the dissociation rate constants, 1-100 s-1. The sugar-binding proteins react at equal rates with the alpha and beta anomeric forms of their substrates. The ligand dissociation rates measured in vitro are consistent with the corresponding Vmax values observed for in vivo active transport. The association rate constant for the L-histidine-binding protein is 5-10 times greater than the corresponding rate constants for the sugar-binding proteins. A similar, large bimolecular rate, approximately 1 X 10(8) M-1 s-1, has been observed for the E. coli L-glutamine-binding protein (Weiner, J. H., and Heppel, L. A. (1971) J. Biol. Chem. 246, 6933-6941) and appears to reflect favorable electrostatic interactions between the charged amino acid and the surface of the protein molecule.

ATP-Binding Cassette Transporters↗
PubMed1983

Proton nuclear magnetic resonance studies of isonitrile-heme protein complexes.

Steric interactions between bound ligand molecules and the valine E11 methyl groups of human hemoglobin and sperm whale myoglobin have been examined directly by high resolution NMR techniques. The methyl proton resonances of this amino acid are shifted markedly upfield and away from the bulk of the protein resonances by the shielding effect of circulating pi electrons in the porphyrin ring. We have monitored the valine resonance in the presence of CO and a series of isonitriles and found considerable shifts in its position, both between the various protein complexes and among the different liganded states. The ring current shifts of the gamma 1-methyl group of Val E11 in the CO forms of isolated alpha and beta chains and myoglobin are -2.70, -2.91, and -3.30 ppm, respectively. In all the proteins, these positions show little change in going from bound CO to bound methyl and ethyl isocyanide. In alpha subunits and myoglobin, n-propyl and n-butyl isocyanide binding produces marked decreases in the magnitude of these shifts, indicating that the valine residue has been forced away from the center of ring by the presence of these large ligand molecules. In the case of beta subunits, however, only tert-butyl isocyanide produces a marked decrease (from -2.91 to -1.99 ppm) in the ring current shift of the valine methyl protons. New peaks were observed in the isonitrile-protein spectra and identified as ligand proton resonances by comparing the spectra of normal and totally deuterated isonitrile complexes. The magnitudes of the ring current shifts for the terminal methyl protons of ethyl isocyanide suggest a linear geometry for the Fe equal to C equal to N - C bonds in beta chains and a bent geometry for alpha chains. The bent geometry in alpha subunits appears to be dictated by the position of the Val E11 methyl group which is located further up from the heme plane but closer to the heme center than the corresponding position of the beta subunit residue. The free energy changes for ethyl isocyanide binding to the two chains are nearly identical, suggesting that the linear and bent geometries are energetically equivalent. Myoglobin ethyl isocyanide complexes exhibit ligand ring current shifts intermediate to those observed for the hemoglobin subunits. Assignment of resonances and positions to the alkyl protons of the longer isonitriles is more difficult.

Animals↗
PubMed1983

The kinetic mechanism of heme binding to human apohemoglobin.

The formation of hemoglobin from free CO-heme and apoprotein is, at minimum, a two-step process. The first reaction involves very rapid equilibration of the heme groups with hydrophobic regions of the globin molecule. The spectrum of the resultant heme-globin complex is analogous to that of CO-heme incorporated into phospholipid membranes. The Soret absorbance maximum of this species is at about 413 nm, which suggests strongly that the iron-histidine bond has not been formed. The equilibrium dissociation constant for the formation of the heme-globin complex is 6.2 +/- 2 microM at pH 7.2 and 10 degrees C. This constant increases with increasing pH, with the addition of inositol hexaphosphate, with modification of the protoporphyrin vinyl groups, and with increasing glycerol concentration. The second step in hemoglobin formation is unimolecular and appears to involve a large conformational transition which results in iron-histidine bond formation and the appearance of a native CO-hemoglobin spectrum. The rate of this process is 500 +/- 150 s-1 at pH 7.2 and 10 degrees C and is little affected by changes in pH, the addition of organic phosphates, or heme modification. Increasing the solvent viscosity to about 10 c.p. by the addition of 50% glycerol causes a 10-fold decrease in the rate of this conformational change. This suggests that large protein movements, presumably folding around the heme, are involved in the final stages of hemoglobin formation. Iron-histidine bond formation does not appear to be a rate-limiting step.

Apoproteins↗
PubMed1983

The oxidation of yeast Complex III. Evidence for a very rapid electron equilibration between cytochrome c1 and the iron-sulfur center.

The reoxidation of reduced cytochrome c1 by potassium ferricyanide follows pseudo-first order kinetics with k = 4 x 10(4) M-1 s-1. However, the reoxidation of this cytochrome in two-electron reduced Complex III does not follow any simple rate law although the overall rate of reaction is essentially unchanged. The observed kinetics can be well fitted with a model in which ferricyanide reacts exclusively with cytochrome c1 together with very rapid electron transfer from the reduced iron-sulfur center to cytochrome c1. Neither removal of coenzyme Q from the complex nor prior incubation with antimycin A had any effect on the observed kinetics of reoxidation.

Chemical Phenomena↗
PubMed1982

Charge transfer complexes between pteridine substrates and the active center molybdenum of xanthine oxidase.

Anaerobic addition of lumazine (2,4-dihydroxypteridine) to xanthine oxidase leads to a rapid bleaching of the enzyme with concomitant formation of a long wave-length species with maximum absorbance at 650 nm. At the conclusion of the reaction there is a net increase in absorbance at wavelengths greater than 550 nm. A spectrally similar species is obtained when violapterin is added to dithionite-reduced enzyme. This long wavelength absorbance is not accompanied by an EPR signal characteristic of flavin neutral radical and it is produced equally well in flavin-free xanthine oxidase. Its production is eliminated by the inhibitors, cyanide and allopurinol, which react at the molybdenum center. We conclude that this new species is a charge transfer complex between Mo(IV) and the product of the reaction, violapterin (2,4,7-trihydroxypteridine). A similar, though less intense, absorbance at 650 nm is also obtained when lumazine is bound to the reduced enzyme. These Mo(IV)-pteridine charge transfer compounds are optically active and exhibit intense circular dichroism centered at 630 nm.

Anaerobiosis↗
PubMed1981

The reaction of reduced xanthine oxidase with oxygen. Kinetics of peroxide and superoxide formation.

Product formation during the oxidation of xanthine oxidase has been examined directly by using cytochrome c peroxidase as a trapping agent for hydrogen peroxide and the reduction of cytochrome c as a measure of superoxide formation. When fully reduced enzyme is mixed with high concentrations of oxygen, 2 molecules of H2O2/flavin are produced rapidly, while 1 molecule of O2-/flavin is produced rapidly and another produced much more slowly. Time courses for superoxide formation and those for the absorbance changes due to enzyme oxidation were fitted successfully to the mechanism proposed earlier (Olson, J. S., Ballou, D. P., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4363-4382). In this scheme, each oxidative step is initiated by the very rapid and reversible formation of an oxygen.FADH2 complex (the apparent KD = 2.2 X 10(-4) M at 20 degrees C, pH 8.3). In the cases of 6- and 4-electron-reduced enzyme, 2 electrons are transferred rapidly (ke = 60 s-1) to generate hydrogen peroxide and partially oxidized xanthine oxidase. In the case of the 2-electron-reduced enzyme, only 1 electron is transferred rapidly and superoxide is produced. The remaining electron remains in the iron-sulfur centers and is removed slowly by a second order process (ks = 1 X 10(4) M-1 s-1). When the pH is decreased from 9.9 to 6.2, both the apparent KD for oxygen binding and the rapid rate of electron transfer are decreased about 20-fold. This result is suggestive of uncompetitive inhibition and implies that proton binding to the enzyme-flavin active site affects primarily the rate of electron transfer, not the formation of the initial oxygen complex.

Animals↗
PubMed1981

Effects of solvent composition and viscosity on the rates of CO binding to heme proteins.

The rate of CO binding to myoglobin increases 4-fold, from 5 X 10(5) M-1 s-1 to 2 X 10(6) M-1 s-1, in going from 0 to 80% glycerol in phosphate buffer at pH 7, 20 degrees C. Under the same conditions, the rate of CO binding to protoheme decreases monotonically from about 1 X 10(8) M-1 s-1 to 2 X 10(7) M-1 s-1. The kinetic behavior of protoheme at neutral pH is that expected for a diffusion-controlled reaction. Increasing solvent viscosity causes a decrease in the observed second order rate constant. In contrast, the behavior of the myoglobin indicates quite clearly that internal, nondiffusive processes are limiting the speed of the reaction. The rate enhancement is due to an increase in the standard chemical potential of the ligand molecule as the polyalcohol concentration is increased. Both types of behavior are observed for ligand binding to protoheme in 0.1 N NaOH; first an increase and then a decrease in rate is observed as the concentration of glycerol is increased. At low glycerol concentrations, the reaction rate is limited by a first order process. At high concentrations, the rate becomes diffusion-controlled and exhibits a dependence on the reciprocal of the solvent viscosity. The data for all these conditions have been analyzed empirically in terms of a single free energy barrier and more specifically in terms of a consecutive reaction scheme.

Animals↗
PubMed1980

Kinetic studies of inducer binding to lac repressor.operator complex.

The rates of binding of inducer molecules to the lactose repressor protein are significantly affected by the presence of bound operator DNA fragments. The association rate is decreased from 4.6 x 10(4) M-1S-1 to 1.0 x 10(4) M-1S-1 by the presence of saturating amounts of operator DNA fragments. The inducer dissociation rate was measured by dilution of repressor . operator . inducer complexes and by displacement of the sugar molecule from repressor by the binding of operator DNA. The value for the dissociation rate in the presence of bound DNA was 0.8 S-1; this is 4-fold greater than the inducer dissociation rate measured in the absence of operator. These kinetic results suggest that repressor should exhibit a 20-fold lower affinity for inducer when operator DNA fragments are bound; this prediction is in agreement with previous equilibrium measurements. Measurement of the rate of operator fragment dissociation from repressor using a nitrocellulose filter assay yields a value of 0.04 S-1 in the absence of inducer; this dissociation rate is too rapid to measure in the presence of bound inducer molecules. Calculations assuming that the DNA-protein association rate is unaffected by sugar binding suggest that the rate of dissociation of DNA from repressor containing 4 bound inducer molecules may be as high as 40 S-1. Determination of the apparent association rate for inducer binding to operator DNA fragments indicates that DNA molecules do dissociate from the protein . operator . inducer complex at intermediate stages during the sugar binding reaction. The exact point at which dissociation occurs at a complex function of the concentrations of operator and inducer and the relationship between the rates for sugar binding and for operator fragment dissociation which are comparable for some of the protein species.

Allosteric Regulation↗
PubMed1980

Equilibrium binding of alkyl isocyanides to human hemoglobin.

The reactions of human hemoglobin with a series of 13 alkyl isocyanides have been examined in equilibrium titration experiments at pH 7, 20 degrees C. The ligands include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, (+)- and (-)-sec-butyl, tert-butyl, n-pentyl, n-hexyl, cyclohexyl, and benzyl isocyanides. All of these compounds exhibit sigmoidal binding curves; however, the amount of cooperativity expressed decreases with ligand length and increases as the alkyl side chains become more highly substituted. The overall affinity of hemoglobin for these compounds also exhibits a complex dependence on ligand size and stereochemistry. These results have been interpreted in terms of competing favorable hydrophobic interactions and unfavorable protein steric effects. The overall chemical potentials of the bound ligands were calculated from the sum of the observed binding free energy change and the relative chemical potentials of the isonitriles in aqueous solution. The resulting values allowed the construction of a rough, three-dimensional free energy map of steric hindrance at the sixth coordination position of the heme iron atom. This scheme suggests a cylindrical cavity of weak protein interactions into which ethyl isocyanide can easily fit. This cavity or mobile region of protein structure is surrounding by a more rigid region which results in large unfavorable steric interactions. Finally, this ring of more rigid structure is followed by an outer area where considerably smaller steric hindrance effects are observed.

Hemoglobins↗
PubMed1980

Kinetic and cooperative mechanisms of ligand binding to hemoglobin.

The reactions of 13 isonitriles with deoxyhemoglobin have been examined and characterized at pH 7, 20 degrees C. Kinetic studies have shown that these ligands can be divided into two mechanistic classes based on their size and stereochemistry. The larger and branched compounds (isopropyl, all butyl isomers, n-pentyl, n-hexyl, cyclohexyl, and benzyl isocyanides) exhibit biphasic time courses at all ligand concentrations as a result of intrinsic differences between the reactivities of the alpha and beta subunits. In contrast, the smaller isonitriles (methyl, ethyl, and n-propyl isocyanides) exhibit monophasic, and often accelerating, time courses at high ligand concentrations. At low concentrations, the smaller isonitriles also exhibit biphasic time courses; however, in this case, the two phases are due to marked differences between the dissociation rate constants of the high and low affinity quaternary conformations of the protein. Sets of equilibrium and kinetic data for the binding of 11 of the isonitriles were fitted to an expanded version of the two-state allosteric model first described by Monad, Wyman, and Changeux (Monod, J., Wyman, J., and Changeux, J.-P. (1965) J. Mol. 12, 108 118). The resultant rate and equilibrium constants for the R (high affinity) and T (low affinity) states were used to calculate chemical potentials for ligand molecules bound to the heme iron and for the kinetic barriers experienced by these compounds during the binding process. For the beta subunits, both the difference between the bound chemical potentials for the R and T states and the differences between the barrier potentials for the two protein conformations are independent of ligand length and stereochemistry. Thus, it would appear that steric interactions are not a major factor in the expression of cooperativity by these subunits. For alpha chains, a 30% decrease in the difference between the bound R and T state potentials is observed in going from methyl to n-hexyl isocyanide. In addition, a marked increase in the height of the T state, alpha chain kinetic barrier is observed with increasing length of the alkyl side chain. Thus, steric hindrance between the bound ligand molecule and protein residues at the sixth coordination position of the heme iron atom does appear to play a significant role in the expression of cooperativity by the alpha subunits, at least for the larger alkyl isocyanides.

Hemoglobins↗