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Biomedical subjects

G H Hoa

Publications and source records attributed to G H Hoa.

9 recordsLinked to original sources
PubMed1992

Substrate analogue induced changes of the CO-stretching mode in the cytochrome P450cam-carbon monoxide complex.

The CO-stretching mode of the carbon monoxide ligand in reduced cytochrome P450cam, in the absence or presence of camphor and in the presence of nine different camphor analogues, was measured at room temperature using Fourier transform infrared spectroscopy. Substrate-free cytochrome P450cam--CO reveals a broad, slightly structured band resulting from an overlap of several stretching mode signals. The multitude of the signals indicates that cytochrome P450 exists in a dynamic equilibrium of several conformational substates. Binding of camphor or camphor analogues strongly influences this equilibrium. For substrate analogues which are not able to form a hydrogen bond to the hydroxyl group of tyrosine 96, the CO-stretching band is rather broad and asymmetric. In contrast, substrate analogues with one quinone group which form a hydrogen bond to the Tyr96 OH induce a shift and a sharpening of the CO-stretching mode band. For substrate analogues with two hetero groups, the infrared spectrum is slightly asymmetric or a minor band appears. Sterical hindrance, substrate mobility, and protein flexibility finally determine the position and width of the CO-stretching mode signals.

Camphor↗
PubMed1992

A critical role of protein-bound water in the catalytic cycle of cytochrome P-450 camphor.

The rates of NADH oxidation during the hydroxylation of camphor by cytochrome P-450cam were followed in the presence of co-solvents used to increase the osmotic pressure surrounding the protein-bound water. As a result, the measured Vmax decreases independently of the perturbant tested. Roughly 28 molecules of water, involved during the catalytic cycle, are deduced from the variation of Vmax as a function of osmotic pressure. These molecules, in part, could be those present in the cytochrome P-450cam-putidaredoxin interface.

Camphor 5-Monooxygenase↗
PubMed1992

High pressure induced inactivation of ferrous cytochrome P-450 LM2 (IIB4) CO complex: evidence for the presence of two conformers in the oligomer.

The effect of high pressure on the spectral properties of cytochrome P-450 LM2(Fe2+)-CO complex was studied. The application of high pressure was shown to induce the conversion of cytochrome P-450 to P-420. In the solution when P-450 was oligomeric only about 65% of the total converted to P-420. The remaining portion of cytochrome P-450 was stable at pressures up to 6 kbar. When P-450 was incorporated into membranes or when it was succinylated, the proportion of the pressure sensitive fraction was slightly higher (about 75%). Dissociation of P-450 oligomers into monomers was made by addition of 0.2% Triton N-101. Monomers were the most sensitive to pressure; they could be completely converted to P-420. These results have been interpreted as evidence for the existence of two different conformers of P-450 LM2, which differ in pressure stability. Splitting between these two states appears to be a result of the oligomeric organization of cytochrome P-450 in solution and in the membrane.

Animals↗
PubMed1992

Cytochrome c and cytochrome c oxidase interactions: the effects of ionic strength and hydrostatic pressure studied with site-specific modifications of cytochrome c.

Seven cytochromes c, in which individual lysines have been modified to the propylthiobimane derivatives, have been prepared. These derivatives were also converted to the porphyrin cytochromes c by treatment with HF. The properties of both types of modified proteins were studied in their reactions with cytochrome c oxidase. The results show that lysines 25, 27, 60, 72, and 87 do not contribute a full charge to the binding interaction with the oxidase. These five residues, with the exception of the lysine-60 derivative, on the front surface of the protein and contain the solvent-accessible edge of the heme prosthetic group. By contrast, lysines 8 and 13 at the top of the front surface do contribute a full charge to the binding interaction with the oxidase. The removal of the positive charge on any one lysine weakens the binding to cytochrome c oxidase by at least 1 kcal (1 cal = 4.1868 J). The presence of bimane at lysines 13 and 87 clearly forces the separation of the cytochrome c and oxidase, but this does not occur with the other complexes. The bimane-modified lysine-13 protein, and to a lesser extent that modified at lysine 8, show the interesting effect of enhanced complex formation with cytochrome c oxidase when subjected to pressure, possibly because of entrapment of water at the newly created interface of the complex. Our observations indicate that the two proteins of the cytochrome c - cytochrome oxidase complex have preferred, but not obligatory, spatial orientations and that interaction occurs without either protein losing significant portions of its hydration shell.

Animals↗
PubMed1976

Implications of electrostatic potentials on ribosomal proteins.

Potentiometric studies of ribosomal particles 30S, 50S, and 70S, were designed to investigate possible implications of the electrostatic potentials developed by the 16S and 23S rRNA fractions. Release of protons and proton titrations of these ribosomal fractions were examined as a function of Mg2+ and K+ concentrations. The effects of these cations fit the polyelectrolyte theory remarkably well and are discussed accordingly.

Bacterial Proteins↗
PubMed1975

The pH dependence of the hydrolysis of benzoyl-L-arginine ethyl ester in cooled mixed solvents.

Tables of protonic activity (paH) of a number of buffers, determined in mixed solvents and at subzero temperatures, are reported for the following media: water-1,2-propanediol, water-glycerol, and water-dimethylsulfoxide (50:50, in volume). These data with those previously reported allowed us to study enzymic reactions under these conditions. The paH dependence of the tryptic hydrolysis of benzoyl-L-arginine ethyl ester has been studied in the presence of organic solvents (methanol, ethylene glycol, 1,2-propanediol, glycerol, and dimethylsulfoxide, all 50% by volume) between 20 and -20 degrees. The results have allowed us to show the validity of our paH scales in mixed solvents. The paH profiles obtained under these conditions are similar to those observed in pure water at 20 degrees. They are shifted nevertheless by both solvent and temperature. Such shifts are interpreted in terms of the effects of solvents and temperature on pKES on the basis of the conclusions drawn from a study of the effect of these variables on small dissociable molecules. The results obtained under these conditions of solvents and temperature are consistent with the presence at the active site of the enzyme of a histidine residue, and thus provide, concerning the solvent effect, a direct verification of the method of Findlay et al. (Findlay, D., Mathias, A. P., and Rabin, B. R. (1962) Biochem. J. 85, 139-144). On the other hand, the large temperature interval provided by the low temperature procedure, allows us to vary significantly the pK of ionizable groups of the enzymes and thus makes possible their identification, on the basis of their enthalpy of ionization.

Arginine↗