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.

Interaction of 13 CO 2 and bicarbonate with human hemoglobin preparations.

Formation of (13)C-resonances attributable to carbamino derivatives has been observed in human erythrocyte hemolysate preparations equilibrated with (13)CO(2) at 33 degrees . Carbamino formation is most marked in deoxyhemoglobin and at alkaline pH, and is very largely inhibited by the addition of 2,3-diphosphoglycerate or conversion to oxyhemoglobin. The prominent carbamino resonance at 30.0 ppm upfield of CS(2) is visible in the spectrum of packed, deoxygenated erythrocytes equilibrated in (13)CO(2). This chemical shift falls close to that observed with sperm-whale myoglobin and within 2 ppm upfield of that seen with simple amino acids and peptides. The bicarbonate-carbonate resonance near 33 ppm is broad in the hemoglobin preparations, which always contain some carbonic anhydrase, but becomes narrow in the presence of the carbonic anhydrase inhibitor, acetazolamide. The nuclear magnetic resonance condition of intermediate exchange rate with dissolved CO(2) (68.4 ppm) obtains in the absence of inhibitor. The process has marked consequences in reducing the spin-lattice relaxation time, T(1), of the bicarbonate resonance by more than 10 times. The deoxyhemoglobin carbamino resonance has a T(1) value of 700 msec, indistinguishable from that of the protein carbonyl resonance envelope.

Complete amino acid sequence of the major component myoglobin from the humpback whale, Megaptera novaeangliae.

The complete primary structure of the major component myoglobin from the humpback whale, Megaptera novaeangliae, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Over 80% of the amino acid sequence was established from the three peptides resulting from the cleavage of the acetimidated apomyoglobin at the three arginine residues with trypsin. The further digestion of the central cyanogen bromide peptide with trypsin and S. aureus strain V8 protease enabled the determination of the remainder of the covalent structure. This myoglobin differs from that of sperm whale, Physeter catodon, at 12 positions, and dwarf sperm whale, Kogia simus, at 14 positions, finback whale Balaenoptera physalus at 3 positions, minke whale, Balaenoptera acutorostrata at 2 positions, and California gray whal Eschrichtius gibbosus, at 1 position. All of the substitutions observed in this sequence fit readily into the three-dimensional structure of sperm whale myoglobin.