Biomedical subjects

D Whitford

Publications and source records attributed to D Whitford.

25 records · Page 2Linked to original sources

PubMed1991

The promotion of self-association of horse-heart cytochrome c by hexametaphosphate anions.

In the presence of the highly charged hexametaphosphate anion, horse heart cytochrome c aggregates to form stable protein complexes. The formation of protein aggregates has been detected by high-resolution 1H-NMR spectroscopy from an increase in the linewidth of resolved ferricytochrome c resonances with hexametaphosphate concentration. Alternatively, analytical ultracentrifugation reveals protein association from the increase in apparent sedimentation coefficients of cytochrome c in the presence of equimolar hexametaphosphate. Protein aggregation is dependent on the concentration of background electrolyte since in the range 10-150 mM sodium cacodylate alternative stabilisation of dimeric and trimeric complexes was observed by both NMR and analytical ultracentrifugation. A model is proposed for the mechanism of protein aggregation caused by polyphosphate binding to the surface of cytochrome c.

Animals↗

PubMed1991

The location of the polyphosphate-binding sites on cytochrome c measured by NMR paramagnetic difference spectroscopy.

Analyses of unimolecular electron self-exchange reactions provide a comparatively simple and direct approach to understanding biological electron transfer. Such studies are currently limited by a lack of well characterised aggregating systems. In the presence of sodium hexametaphosphate, cytochrome c forms stable protein aggregates as a result of binding hexametaphosphate at a single site on its surface (preceding paper in this issue of the journal). Here we report the location of the principal polyphosphate binding site on the surface of cytochrome c for both hexametaphosphate and a second polyphosphate, tripolyphosphate determined using 1H-NMR spectroscopy in conjunction with the relaxation probe potassium hexacyanochromium(III). Addition of either hexametaphosphate or tripolyphosphate to ferricytochrome c in the presence of the relaxation probe causes a decrease in intensity of several resonances in the paramagnetic difference spectrum, including Phe82 ortho/meta, Ile85 delta methyl and Ile9 gamma methyl. Together these effects put the site of polyphosphate binding close to lysines 13, 86, and 87. Additionally the effect of sodium tripolyphosphate and sodium trimetaphosphate on cytochrome c aggregation is described. The potential role of this site in anion-induced cytochrome c aggregation is discussed.

Animals↗

PubMed1990

The formation of protein complexes between ferricytochrome b5 and ferricytochrome c studied using high-resolution 1H-NMR spectroscopy.

The association of the tryptic fragment of bovine microsomal cytochrome b5 with cytochrome c has been studied by one- and two-dimensional 1H-NMR spectroscopy. The association of cytochromes to form protein complexes is apparent from the increase in linewidths for resonances of ferricytochrome b5 as well as small perturbations in their chemical shifts that occur upon increasing the cytochrome c/b5 molar ratio. The changes in the chemical shifts of hyperfine shifted resonances of ferricytochrome b5 with increasing ratios of ferricytochrome c indicate the formation of binary 1:1 complexes and ternary 1:2 complexes. Similarly, titrations of the linewidth of resolved resonances of ferricytochrome b5 are consistent with stoichiometries of 1:1 and 1:2 for complexes formed between cytochromes b5 and c. Surprisingly, in the 1:1 complex, mobility is shown to be a function of ionic strength. Two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY) of the binary complex formed between ferricytochrome b5 and c indicate that the positions of many resonances attributable to amino acids are unaltered by protein association, although distinctive chemical shift changes are detected in the alpha-CH of the haem C17 propionate. The protein complex detected by NMR is discussed with respect to the model for the binary complex proposed by Salemme and possible mechanisms of electron transfer.

Animals↗

PubMed1990

An analysis of pseudocontact shifts and their relationship to structural features of the redox states of cytochrome b5.

The assignment of proton resonances in both redox states of a heme protein is necessary for the evaluation of pseudocontact shift data. Many new assignments are presented here for cytochrome b5, particularly in the paramagnetic oxidised state, thereby allowing both the calculation of electronic g-tensor values with the magnetic axis orientation and a comparison of observed and calculated pseudocontact shifts utilising a computational procedure. The possible redox linked conformational changes are found to be minimal in contrast with cytochrome c although the procedure additionally highlights aspects of the mobility of certain residues in cytochrome b5. In this respect the residue Gly-42 appears mobile both by this method and by the observation from NMR spectra of a major and minor conformation in this region.

Amino Acid Sequence↗

PubMed1988

Investigation of the solution structures and mobility of oxidised and reduced cytochrome b5 by 2D NMR spectroscopy.

Two-dimensional 1H NMR spectroscopy is used to examine the structure and mobility of cytochrome b5 in solution. The assignment of many residues and the interpretation of nuclear Overhauser effects (NOEs) in both redox states allow definition of secondary structural elements. Comparison with X-ray diffraction data shows that differences between crystal and solution structures are small. The dynamics of the protein are examined and the protein is shown to be more mobile than cytochrome c. The relationship of the structure and dynamics to the electron transfer function of cytochrome b5 is discussed.

Animals↗

PubMed1986

The modulation of cytochrome c electron self-exchange by site-specific chemical modification and anion binding.

The site-specific chemical modification of horse heart cytochrome c at Lys-13 and -72 using 4-chloro-3,5-dinitrobenzoic acid (CDNB) increases the electron self-exchange rate of the protein. In the presence of 0.24 M cacodylate (pH* 7.0) the electron self-exchange rate constants, kex, measured by a 1H NMR saturation transfer method at 300 K, are 600, 6 X 10(3) and 6 X 10(4) M-1 X s-1 for native, CDNP-K13 and CDNP-K72 cytochromes c respectively. Repulsive electrostatic interactions, which inhibit cytochrome c electron self-exchange, are differentially affected by modification. Measurements of 1H NMR line broadening observed with partially oxidised samples of native cytochrome c show that ATP and the redox inert multivalent anion Co(CN)3-6 catalyse electron self-exchange. At saturation a limiting value of approximately 1.4 X 10(5) M-1 X s-1 is observed for both anions.

Adenosine Triphosphate↗

PubMedDate not supplied

The measurement of changes in pH associated with electrochemically driven respiration in rat liver mitochondria.

Using an optically transparant thin layer electrode, it has been possible to measure the pH changes associated with the electrochemical turnover of horse heart cytochrome c in the presence of rat liver mitochondria and oxygen. Direct electrochemistry of cytochrome c at a gold electrode modified with bis(4-pyridyl)bisulfide allowed electron flux (current) to be measured simultaneously with the differential change in absorbance associated with phenol red, a pH-sensitive dye. Although the alkalinization due to the reduction of oxygen to water was readily observed, any initial acidification associated with proton pumping was not detected. It is suggested that at the high ratios of oxidized-to-reduced cytochrome c present during the steady state attained, proton pumping may be absent or more localized.

Animals↗