Yasunori Enoki

Oxygen equilibria of half cyanomet hybrids of human and canine haemoglobins

Abstract By use of acid hybridization techniques, special hybrid molecules were produced between human adult and canine haemoglobins, in which either one of the species of the constituent subunits was fixed specifically in cyanomet form and consequently deprived of its oxygenation function. These molecules were characterized by increased affinity toward oxygen and complete absence of haemhaem interaction (Hills exponent = 1.0). The latter finding is clearly in contrast with the higher n -value (1.9 or so) of human haemoglobin randomly oxidized by 50%. The bulk of the oxygen Bohr effect on the alkaline side was well preserved although its extent was more or less reduced. The molecules of comparable subunit composition, irrespective of which species of subunit was inactivated, were found to have comparable behaviour in their general oxygenation properties. Similar results were also obtained in the oxidation kinetics of the hybrids with ferricyanide at pH 5.9. Determination of the reactive SH groups of the hybrid haemoglobins suggested that both α- and β-subunits in canine haemoglobin might have one reactive and one unreactive SH group per chain.

Properties of isolated active γF subunits of foetal haemoglobin

Abstract Physiologically active γ F chains were isolated from foetal haemoglobin by our differential mercuration procedure, a modification of the method of Bucci & Fronticelli (1965), and their properties were studied. Identification of the subunits was by gel electrophoresis, tryptic peptide mapping and on the basis of the well-resolved tryptophan fine-structure absorption band in the ultraviolet. Sedimentation analysis showed that the isolated subunits existed in a tetrameric state. The number of the reactive SH groups was estimated as one per chain, which shows that all the SH groups of the chain are fully reactive. The oxygen equilibrium was characterized by an increased affinity toward oxygen, and absence of haem-haem interaction and the Bohr effect. The oxygen affinity was found to be lower than that of the β A subunit. These findings are essentially the same as those for other single subunit haemoglobins. The subunit was less resistant to alkaline denaturation in the deoxy form than in the oxy form, and the same was also found in foetal haemoglobin.

Cleavage at the α1β1 contact and its effect on the oxygen equilibrium of human haemoglobin

The molecular weight of human oxyhaemoglobin in 1.0 m-potassium iodide is reduced to between 34,000 and 36,700 at protein concentrations between 0.15 and 0.73 per cent, which shows that the haemoglobin is almost wholly dimeric in this solvent. However, deoxygenation causes the molecular weight to return to about 60,000, which shows that re-association from dimer to tetramer occurs. Hybridization between human and canine oxyhaemoglobins in 1.0 m-KI occurs readily to produce new hybrid species. No hybrid molecules are produced when the haemoglobins are deoxygenated. The association-dissociation equilibria in 1.0 m-KI are therefore believed to be tetramer ⇌ dimer ⇌ monomer in the liganded form, and to be tetramer ⇌ dimer in the non.liganded form. The initial dissociation into dimer appears to be symmetric. n nThe value of n in Hills equation decreases by 1 in 1.0 to 1.5 m-KI. This decrease in n parallels the decrease in molecular weight and the increase in hybrid formation; in contrast, the n value in 2.0 m-NaCl does not decrease. Therefore, it seems probable that the decrease in co-operativity by 1 in 1.0 to 1.5 m-KI results from weakened subunit interactions at the α1β1 contact during oxygenation. n nThis means that for the expression of full co.operativity both the contact surfaces, α1β1 and α1β2, are important. The oxygen affinity is reduced to a minimum at 0.3 m-KI, and increased at the lower and higher concentrations of KI. The acid and alkaline Bohr effects are 0.45 and −0.34, respectively, at 16 °C in 1.0 m-KI in the ranges of pH from 6.2 to 6.4 and 7.2 to 8.0. These values are not changed by temperature. The apparent enthalpy change for the oxygenation reaction is 11.4 kcal/mol in both the presence and absence of KI.

Oxygen transport and phosphorylated glycolytic intermediates of the ruminant blood—I. sika deer (Cervus nippon nippon)

Abstract 1. 1. Values for several hematologic parameters were determined for sika deer ( Cervus nippon nippon ), the most prominent features of which were a high red cell count, (10·56±2·64) × 10 6 /μl, and a low MCV, 39·3 ± 6· μ 3 . 2. 2. The hemoglobin of sika deer was a single component of the same mobility when examined by starch gel electrophoresis. 3. 3. The oxygen affinity of deer blood was slightly lower than that of human adult blood through the physiological pH range (pH 6·9 ∼ 7·7). 4. 4. There was no significant difference in the intra- vs extra-cellular pH relationship between deer and human adult blood. 5. 5. Concentrations of 2,3-diphosphoglycerate (2,3-DPG) and the other organic phosphates were very low in deer red cells. 6. 6. It is concluded that the lower oxygen affinity of deer blood in the essential absence of co-factor phosphates such as 2,3-DPG might be of an intrinsic nature reflecting the characteristic primary structure of the deer hemoglobin itself.

An evidence for dissociation of haemoglobin into monomer in concentrated potassium iodide.

Abstract Most of haemoglobin in 1.0 m -KI at neutral pH is in a state of dimer, which is also in rapid equilibrium with monomer. Such a mode of dissociation appears to differ from those in a concentrated NaCl solution, in which most of haemoglobin remains solely in a dimeric state. Hybridization is an extremely sensitive technique for detecting monomer since hybrids may be formed through the dissociation of haemoglobin into monomer.