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Biomedical subjects

F R Gurd

Publications and source records attributed to F R Gurd.

At least 73 records · Page 4Linked to original sources
PubMed1976

Nuclear magnetic resonance studies of sperm whale myoglobin specifically enriched with 13C in the methionine methyl groups.

The Cepsilon methyl group of the 2 methionine residues in sperm whale myoglobin was enriched with respect to 13C. This was accomplished by treatment of the apomyoglobin at pH 4 at room temperature with a 100-fold proportion of 13CH3I to form an intermediate containing enriched S-methylmethionine. Unselective demethylation to regain the apomyoglobin structure was accomplished by treatment at pH 10.5 with 0.5 M dithioerythritol at 37 degrees for 18 h. Reagents were removed at each stage by dialysis against dilute sodium azide solution. Hemin was reincorporated to form the holoprotein in a way that avoided the presence of an excess of the small molecule. After chromatographic purification the enriched myoglobin was obtained in a yield of between 29 and 60%. The composition, absorbance spectrum, circular dichroism spectrum, isoionic point, electrophoretic behavior, and oxygen-binding behavior following reduction were all indistinguishable from those of the virgin protein. NMR measurements were made at 15.1, 25.2, and 67.9 MHz at 27-30 degrees. The two enriched loci are represented by separate resonances that appear slightly downfield of the spectral position of the corresponding resonance in free methionine. The positions of these resonances are sensitive to pH and to the ligand bound at the heme group which is approximately 17 A distant from each methionine Cepsilon. On the basis of two separate types of experiment the downfield resonance was assigned to methionine 55 and the upfield resonance to methionine 131. Part of the observed variations in chemical shift could be treated as arising from pseudocontact interactions but part was ascribed to structural changes communicated to the environment of each methionine residue as a result of changes in heme ligand, pH, or temperature. The linewidths of the methionine Cepsilon resonances are narrowed by increasing temperature according to an Arrhenius energy of activation of nearly 3 kcal. The spin-lattice relaxation times, T1, of the two methionine Cepsilon resonances at the three spectrometer frequencies were interpreted to indicate the existence of rotational motions in each side chain in addition to that about the Sdelta-Cepsilon bond. The results as a whole show that the two methionine side chains undergo continuous variations in environment, and that these variations are controlled by events at a distance within the protein structure. It is suggested that the structural lability serves the function of facilitating conformational variations and adjustments within the heme pocket.

Amino Acids↗
PubMed1976

Complete amino acid sequence of the myoglobin from the Atlantic bottlenosed dolphin, Tursiops truncatus.

The complete amino acid sequence of the major component myoglobin from the Atlantic bottlenosed dolphin, Tursiops truncatus, was determined by specific cleavage of the protein to obtain large peptides that are readily degraded by the automatic sequencer. Three easily separable peptides were obtained by cleaving the protein with cyanogen bromide at the 2 methionine residues and 4 peptides were obtained by cleaving the methyl acetimidated protein with trypsin at the 3 arginine residues. By subjecting 4 of these peptides and the apomyoglobin to automatic Edman degradation, over 80% of the covalent structure of the protein was obtained. The remainder of the primary structure was determined by further digestion of the central cyanogen bromide peptide with trypsin and staphylococcal protease. This myoglobin differs from that of the sperm whale, Physter catodon, at 15 positions, from that of the California gray whale, Eschrichtius gibbosus, at 14 positions, from that of the common porpoise, Phocoena phocoena, at 6 positions, and from the myoglobin of the Black Sea dolphin, Delphinus delphis and the Amazon River dolphin, Inia goeffrensis, at 5 and 7 positions, respecitvely. All substitutions observed in this sequence fit easily into the tertiary structure of sperm whale myoglobin.

Amino Acid Sequence↗
PubMed1976

Complete primary structure of the major component myoglobin of California gray whale (Eschrichtius gibbosus).

The complete primary structure of the major component myoglobin from the California gray whale, Eschrichtius gibbosus, was determined by specific cleavage of the protein to obtain large peptides for degradation by the automatic sequenator. Cleavage at the two methionine residues of the apomyoglobin with cyanogen bromide and at the three arginine residues of the methyl acetimidated protein with trypsin resulted in three and four easily separable peptides, respectively, which when sequenced accounted for 85% of the primary structure. The remainder of the covalent structure was obtained by further digestion of the central cyanogen bromide peptide with trypsin and S. aureus strain V8 protease. This protein differs from that of the sperm whale, Physeter catodon, at 12 positions, from that of the common porpoise, Phocoena phocoena, and the Black Sea dolphin, Delphinus delphis, at 14 positions, and from that of the Amazon River dolphin, Inia geoffrensis, at 7 positions. All substitutions observed in this sequence fit easily into the tertiary structure of sperm whale myoglobin.

Amino Acid Sequence↗
PubMed1976

Carbon 13 resonances of 13CO2 carbamino adducts of alpha and beta chains in human adult hemoglobin.

The principal component of normal adult human hemoglobin Ao, was equilibrated under various conditions with 13CO2. In addition, derivatives containing specifically carbamylated NH2-terinal groups in alpha or beta chains, or both, were prepared by treatment with cyanate, and equilibrated likewise to allow the identification of specific resonances observed by 13C nuclear magnetic resonance. In deoxyhemoglobin, a resonanance at 29.2 ppm upfield of external CS2 was assigned to the alpha chain terminal adduct, and one at 29.8 ppm to the beta chain terminal adduct. In the liganded state as the CO derivative, the terminal adduct on both chains showed a common resonance position at 29.8 ppm. Small effects of pH on the resonance positions were observed. Under certain conditions, a resonance was observed at 33.4 ppm, probably not ascribable to a carbamino compound. A carbamino resonance that became prominent at higher pH was found at 28.4 ppm, and is tentatively ascribed to one or more adducts on epsilon amino groups. The beta chain resonances in particular are minimized by the presence of inositol hexaphosphate or 2,3-diphosphoglycerate. Quantitative analysis of the resonance intensities shows that the effects of conversion from the deoxy to the liganded state in reducing the degree of carbamino adduct is much more pronounced for the beta than for the alpha chains.

Adult↗
PubMed1975

The complete amino acid sequence of the major component myoglobin of Amazon river dolphin (Inia geoffrensis).

The complete amino acid sequence of the major component myoglobin from Amazon River dolphin, Inia geoffrensis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Three easily separable peptides were obtained by cleaving the protein with cyanogen bromide at the methionine residues and four peptides were obtained by cleaving the methyl-acetimidated protein with trypsin at the arginine residues. From these peptides over 85% of the sequence was completed. The remainder of the sequence was obtained by fragmentation of the large cyanogen bromide peptide with trypsin. This protein differs from that of the common porpoise, Phocoena phocoena, at seven positions, from that of the common dolphin, Delphinus delphis, at 11 positions, and from that of the sperm whale, Physeter catodon, at 15 positions. By comparison of this sequence with the three-dimensional structure of sperm whale myoglobin it appears that those residues close to the heme group are most conserved followed by those in nonhelical regions and lastly by those in the helical segments. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.

Amino Acid Sequence↗
PubMed1975

Deuterium nuclear magnetic resonance of deuterium-labeled diacetyldeuterohemin incorporated into sperm whale myoglobin.

The heme derivative 2,4-diacetyldeuterohemin deuterated in the methyl groups of the acetyl moieties was reconstituted with sperm whale apomyoglobin and the two labeled methyl groups in the protein environment were observed by deuterium nuclear magnetic resonance spectroscopy. The results were compared to the free hemin form as the dimethyl ester in chloroform and in a pyridine-water mixture, as well as in the zinc complex form. Under most conditions the two methyl resonances overlie each other to a large degree. Resonance width at half-height is of the order of 25 Hz for the protein and approximately one-third as much for the free hemin at 16 degrees and is little affected by conversion to paramagnetic derivatives. Chemical shifts for the oxy- and carbonmonoxymyoglobins are very similar. In cyanoferrimyoglobin a positive pseudo-contact contribution of 3.04 ppm was computed to explain a relative upfield shift offset in part by a small negative contact shift contribution. The cyanoferrimyoglobin resonance was sensitive to the presence of phosphate buffer as well as to cyclopropane. The aquoferrimyoglobin form shows distinct resonances for the two methyl groups, with the downfield resonance considerably broadened. The expected effects of temperature on chemical shift were observed, the paramagnetic derivatives showing an effect and carbonmonoxymyoglobin showing none. The relaxation behavior was gauged from the line widths and from measurements of spin-lattice relaxation time, T1. The effective rotational correlation time is of the order of 50 ps for the liganded myoglobin forms. The temperature dependence of the line widths may imply an increased retational freedom with increasing temperature. The broadening observed in the aquoferrimyoglobin case is indicative of restricted internal rotational motion of one of the methyl groups. The method is suitable for probing the more mobile structures in proteins and retains its value in the neighborhood of paramagnetic centers.

Animals↗
PubMed1975

Determination of the pK values for the alpha-amino groups of human hemoglobin.

The rate of reaction between alpha-amino groups and cyanic acid was followed at 26 degrees and ionic strength 0.2 M as a function of pH of human hemoglobin Ao solutions to determine the pK and the pH-independent second order rate constant, kappa, for these groups in the alpha and beta chains. At a given point in time, the extent of the reaction was determined by employing the Beckmann Sequencer as a quantitative tool in which the yields of leucine and histidine in the second Edman degradation cycle were used to define the rates of reaction of the alpha and beta chains, respectively. From these results, the individual were evaluated (Garner, M.H., Garner, W.H., and Gurd, F. R.N. (1973) J. Biol. Chem. 248, 5451-5455). Values for pK for the alpha and beta chains were, respectively, 6.74 and 6.93 for cyanoferrihemoglobin, 6.95 and 7.05 for carboxyhemoglobin, and 7.79 and 6.84 for deoxyhemoglobin. Values for kappa, M- minus 1 S-minus 1, for the alpha and beta chains were, respectively, 12.5 and 17 for cyanoferrihemoglobin, 12 and 18 for carboxyhemoglobin, and 91 and 24 for deoxyhemoglobin. Limits of significance were estimated for both variables in each case. The pK results for valine 1alpha agree well with the value obtained by Hill and Davis (1967) J. Biol. Chem. 242, 2005-2012) for carboxyhemoglobin and with that of Kilmartin and Rossi-Bernardi ((1971) Biochem. J. 124, 31-45) for deoxyhemoglobin. Values obtained for sperm whale myoglobin were 7.77 for pK and 7.4 for kappa. The results are useful for the interpretation of the allosteric interactions of hemoglobin with hydrogen ions, with CO2, and with phosphate.

Amines↗
PubMed1975

Electrostatic effects in myoglobin. Application of the modified Tanford-Kirkwood theory to myoglobins from horse, California grey whale, harbor seal, and California sea lion.

The modified Tanford-Kirkwood electrostatic theory (Shire et al., 1974a) was applied to ferrimyoglobins from the following animal species: sperm whale (Physeter catodon), horse, California grey whale (Eschrichtius gibbosus), harbor seal (Phoca vitulina), and California sea lion (Zalophus californianus). Computations were made of the overall hydrogen ion titration curves of the proteins, and of pH and ionic strength variations of ionization equilibria for individual groups in the protein, with particular reference to the hemic acid ionization of the iron bound water molecule. Coordinates and static solvent accessibility were estimated in terms of the sperm whale myoglobin structure. Where possible, theoretical results and experimental data are compared. Some comparative features of charge and ionization properties among the various myoglobins are presented.

Amino Acids↗
PubMed1975

Rotational motions in myoglobin assessed by carbon 13 relaxation measurements at two magnetic field strengths.

Proton-decoupled Fourier transform nuclear magnetic resonance spectroscopy of natural abundance 13C was used to obtain spectra of cyanoferrimyoglobin of sperm whale (Physeter catadon) at 14.1 and 23.5 kG. Comparison of the spin lattice relaxation times at these two field strengths allowed the unambiguous assignment of a rotational correlation time of 22 plus or minus 5 ns for the alpha carbon resonances. The spin lattice relaxation time value for a major band attributable to aromatic carbon atoms also corresponded to a single correlation time, attributable to over-all tumbling of the molecule. Certain narrower resonances reflect other modes of rotational motion in addition to the over-all tumbling. Observations of nuclear Overhauser enhancement and line widths accord with these conslusions.

Animals↗