The effect of urea on the oxygen affinity of sickle cells and normal cells was studied up to a concentration of about 1.0 M. Besides the increase in oxygen affinity found in both normal and sickle cells there was a further increase found only in the sickle cells. This specific increase was caused by the direct inhibition of the polymerization of deoxygenated Hb-S by the urea and was used to measure the extent of this inhibition. Even at concentrations of 1.0 M the urea did not fully inhibit the polymerization. At the urea concentrations recommended for treatment of sickle cell crises and for the oral treatment of the disease there was only a very slight inhibition of polymerization. The small increase in oxygen affinity brought about by these concentrations of urea (equivalent to a log P50 change of 0.016) will cause some additional minimal inhibition of sickling at physiological partial pressures of oxygen. Oxygen dissociation measurements of Hb-A in dilute solution showed that 0.95 M urea had no effect on the interaction of 2,3-DPG and chloride with haemoglobin nor on the haem-haem interactions. This implies that the quaternary 'deoxy' form of the haemoglobin may still form and that the action of urea in inhibiting the polymerization is through interference with the intermolecular bonds rather than by inducing a conformational change which disrupts the bonding. Unless urea at the concentrations recommended for treatment inhibits the sickling of the cells by some mechanism other than its effect on the haemoglobin (such as by an effect on the membrane), these results suggest that it is of little use for the treatment of sickle cell disease.

Anemia, Sickle Cell↗

PubMed1975

The oxygen affinity of haemoglobin E.

Oxygen dissociation studies were carried out on haemoglobin E (Hb E) at both high and low haemoglobin concentrations. Oxygen affinities of fresh red cells from three people homozygous for Hb E and from one with Hb E-beta thalassaemia (Hb-E trait/beta-thal trait) were low in three out of four patients studied, while the oxygen affinity of red cells from an individual with Hb-E was normal 2,3-DPG concentration in the fresh cells from the people with homozygous Hb E or Hb-E trait/beta-thal trait which showed low oxygen affinities were elevated sufficiently to account for the shifts observed. When the cells from two of these people with homozygous Hb E were depleted of 2,3-DPG. their oxygen affinities became the same as that of similarly treated normal cells. Pure 'stripped' Hb E in dilute solution behaved identically to Hb A in respect of P50, Bohr shift, haem-haem interaction, and interaction with inorganic phosphate or 2,3-DPG. Hb E, therefore, has the same oxypgen dissociation properties as Hb A both in dilute solution and in the red cell. The low oxygen affinities found in the fresh cells and in whole blood are caused by high 2,3-DPG concentrations within the cell.

Erythrocytes↗

PubMed1975

The oxygen affinity of haemoglobin Hammersmith.

Oxygen dissociation studies were carried out on red cells and lysates from a patient heterozygous for Hb Hammersmith. The oxygen affinity of the cells at pH 7.1 was decreased, partly by an increased cellular concentration of 2,3-DPG and more importantly by an intrinsic low affinity of Hb Hammersmity. Haem-haem interactions were reduced and an abnormal Bohr effect (pH 7.1-7.4) was found in the cells which would bring about an additional decrease in oxygen affinity at physiological pH. Oxygen dissociation studies on the lysates showed a low oxygen affinity, a normal Bohr effect at 50% saturation, slightly decreased haem-haem interactions and a normal interaction with 2,3-DPG. Estimation of the percentage of Hb Hammersmith in the lysates of the patient's cells by selective precipitation of the abnormal beta chain with p-chloromercuribenzoate (PCMB) showed the presence of 30-33% abnormal haemoglobin. Assuming no interaction between the Hb A and the Hb Hammersmith, oxygen dissociation curves were calculated for Hb Hammersmith alone and these indicated that it has greatly decreased oxygen affinity, a normal Bohr effect, normal 2,3-DPG interaction and (somewhat) decreased haem-haem interactions.

Adult↗

PubMed1975

The concentration dependence of the oxygen affinity of haemoglobin S.

The effect of the concentration of haemoglobin S (Hb S) on its oxygen-dissociation properties was studied using either reconstituted Hb-S cells of different mean corpuscular haemoglobin concentrations (MCHCs) prepared by osmotic lysis, or cells in which Hb S is diluted by the presence of another haemoglobin. Only 4% (phosphate buffer) and 21%(bis Tris) of the low oxygen affinity of fresh Hb-S cells was found to be due to their slightly elevated intracellular 2,3-DPG concentrations since when the cells were depleted of 2,3-DPG most of the low affinity remained. The low affinity showed a marked dependence upon haemoglobin concentration which was absent for 2,3-DPG-depleted Hb-A cells and, by extrapolation, the MCHC at which the oxygen affinities of the Hb-S cells became identical to that of the Hb-A cells was 14.5 g/dl in phosphate buffer and 13.1 g/dl in bis Tris. Both fresh and 2,3-DPG-depleted cells containing another haemoglobin as well as Hb S (Hb-SA, Hb-SC and Hb-SF cells) were also found to have low oxygen affinities provided that the intracellular Hb-S concentration(MC(Hb-S)C) was above a certain level. These also showed a strong dependence upon the MC(Hb-S)C. The mean MC(Hb-S)C at which the low oxygen affinities of the DPG-depleted cells were abolished were 8.3 g/dl (phosphate) and 11.2 g/dl (bis Tris). Hb F in fresh Hb-SF cells brought about a much greater increase in oxygen affinity than the same amount of either Hb A or Hb C. In 2,3-DPG depleted cells Hb A showed a greater ability to 'dilute' the Hb S than did Hb C. The conditions for the low oxygen affinity of Hb S were therefore found to be very similar to those required for the gelling of both pure Hb S, and Hb S in haemoglobin mixtures. It was concluded therefore that the low oxygen affinity of the Hb S was caused by the polymerization and that the difference between the oxygen affinities of Hb-S and Hb-A cells may be used as a measure of the polymerization process.

Erythrocytes↗

PubMed1975