Biomedical subjects

M B Hayes

Publications and source records attributed to M B Hayes.

5 recordsLinked to original sources

PubMed1979

Heat-induced fragmentation of human alpha 2-macroglobulin.

Previous studies have demonstrated that human plasma alpha 2-macroglobulin (alpha 2 M) possesses a single subunit chain (Mr approximately 185,000) when incubated with dodecyl sulfate and dithiothreitol at 37 degrees C and analyzed by dodecyl sulfate-gel electrophoresis. The present study details the observation that heating alpha 2 M to 90 degrees C under identical conditions produces at least two additional polypeptide chains, termed bands II and III, with apparent molecular weights of 125,00 and 62,000. The generation of these fragments is enhanced by increasing the time of incubation. The appearance of band II composition of the buffer, dodecyl sulfate concentrations, or alpha 2 M protein concentration in the incubation mixture. The electrophoretic bands II and III of alpha 2 M have dissimilar 125I-labeled tryptic peptide digests and also differ in their amino acid composition. The heat-induced fragmentation of alpha 2M is not affected by the inclusion of a variety of low molecular weight protease inhibitors, suggesting that the appearance of bands II and III is not due to enzyme-catalyzed hydrolysis. When the subunit chain of alpha 2M is first cleaved by trypsin into the previously described Mr = 85,000 derivative, neither band II nor III material, nor other lower molecular weight products are generated by heat treatment. Furthermore, preincubation of alpha 2M with methylamine prevents fragmentation of the subunit chain. These results indicate that these fragments are neither pre-existing subunits of alpha 2M nor derivatives formed prior to treatment for gel analysis. These data provide evidence that a covalent bond in the alpha 2M molecule is unusually susceptible to heat-induced cleavage.

Amino Acids↗

PubMed1979

Near-heme histidine residues of deoxy- and oxymyoglobins.

Proton NMR titration curves of the histidine Cepsilon-H resonances of the deoxy and oxy forms of human, horse, and sperm whale myoglobins (Mb) were determined and compared with the results for the met and azide forms. One extra titrating resonance (H-8) was observed for each deoxy-Mb compared with the corresponding met-Mb, and a further extra resonance (H-9) was observed for the oxy-Mb form. These resonances correspond to the two additional resonances previously described for azide-Mb [Hayes, M., Hagenmaier, H., & Cohen, J. S. (1975) J. Biol. Chem. 250, 7461--7472]. This new evidence prompts us to reassign these resonances to the near-heme histidine residues.

Heme↗

PubMed1976

Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease.

One of the four titrating histidine ring C-2 proton resonances of bovine pancreatic ribonuclease has been assigned to histidine residue 12. This was accomplished by a direct comparison of the rate of tritium incorporation into position C-2 of histidine 12 of S-peptide (residues 1 to 20) derived from ribonuclease S, with the rates of deuterium exchange of the four histidine C-2 proton resonances of ribonuclease S under the same experimental conditions. The same assignment was obtained by a comparison of the NMR titration curves of ribonuclease S, the noncovalent complex of S-peptide and S-protein (residues 21 to 124) with the results for the recombined complex in which position C-2 of histidine 12 was fully deuterated. The second active site histidine resonance was assigned to histidine residue 119 by consideration of the NMR titration results fro carboxymethylated histidines and 1-carboxymethylhistidine 119 ribonuclease. This assignment is a reversal of that originally reported, and has important implications for the interpretation of NMR titration data of ribonuclease.

Binding Sites↗

PubMed1975

Nuclear magnetic resonance titration curves of histidine ring protons. The effect of temperature on ribonuclease A.

The ionization constants of 3 of the histidine residues of ribonuclease A have beenobtained at 5 temperatures from the nuclear magnetic resonance titration curves of the imidazole C2 proton resonances. Thermodynamic parameters derived from the ionization constants indicate that histidine residues 105 and 119 are fairly well exposed to solvent, while histidine residue 12 is in a somewhat more restricted environment. Measurements of the low pH inflection present in the titration curve of histidine-12 yield a large negative entropy value, indicating that the group givine rise to this inflection is also buried.

Calorimetry↗

PubMed1975

Nuclear magnetic resonance titration curves of histidine ring protons. Human metmyoglobin and the effects of azide on human, horse, and sperm whale metmyoglobins.

Four titrating histidine ring C2 and C4 proton resonances are observed in 220 MHz proton NMR spectra of human metmyoglobin as a function of pH. Values of ionization constants determined from the NMR titration data using an equation describing a simple proton association-dissociation equilibrium are curves (1) 6.6, (2) 7.0, (3) 5.8, and (4) 7.4. Four histidine residues have also been found to be solvent-accessible in human metmyoglobin by carboxymethylation studies (Harris, C.M., and Hill, R.L. (1969) J. Biol. Chem. 244, 2195-2203). Two of the titration curves (3 and 4) deviate significantly from the chemical shift values normally observed for histidine C2 proton resonances. Curve 3, with a low pKa, is shifted downfield at high values of pH and also exhibits a second minor inflection with a pKa value of 8.8. On the other hand, the high pKa curve, 4, is shifted upfield at all values of pH. The characteristics of the NMR titration curves with the lowest and highest pKa values (3 and4) are very similar to curves observed previously with sperm whale and horse metmyoglobins (Cohen, J.S., Hagenmaier, H., Pollard, H., and Schechter, A.N. (1972) J. Mol. Biol. 71, 513-519). These results indicate that the histidine residues from which these curves are derived have unusual and characteristic environments in this series of homologous proteins. The NMR spectra of all three metmyoglobins are changed extensively as a result of azide ion binding, indicating conformational changes affecting the environments of several imidazole side chains. The presence of azide ion causes a selective downfield chemical shift for the low pKa curve and a selective upfield chemical shift for the high pKa curve in all three proteins. Azide also abolishes the second inflection seen in the low pKa curve at high pH. In addition to these effects, the presence of azide ion permits the observation of two additional titrating proton resonances for all three metmyoglobins. Increasing the azide to protein ratio at several fixed values of pH yields results which show that a slow exchange process is occurring with each of the metmyoglobins. In the azide titration studies the maximum changes in the NMR spectra occurred at approximately equimolar concentrations. The NMR results for these proteins in the absence and presence of azide ion are related to x-ray crystallographic studies of sperm whale metmyoglobin and the known alkylation properties of the histidine residues. Tentative assignments of the titrating resonances observed are suggested.

Animals↗