Oxygen affinity and electrolyte distribution of human blood: changes induced by propranolol.

Propranolol causes a massive leakage of potassium ions from red cells, which results in an alteration of the Gibbs-Donnan equilibrium across the red cell membrane. According to such a mechanism, the presence of propranolol significantly increases the hydrogen ion activity of the interior of the red cell, causing a decreased oxygen affinity of hemoglobin according to the classical Bohr effect. No release of 2,3-diphosphoglycerate which may be bound to the membrane is thus necessary to explain the effect of propranolol on the oxygen dissociation curve of blood.

What the intermediate compounds in ligand binding to hemoglobin tell about the mechanism of cooperativity.

The populations of the intermediates in concentrated solutions of hemoglobin A0 equilibrated at various PCO values, pH 7.0, 0.1 M KCl, and 20 degrees C, have been determined using cryogenic methods. Data on CO saturations and distributions of intermediates were analysed in terms of the free energies of dimer-tetramer assembly of the intermediates (G.K. Ackers and F.R. Smith, Annu. Rev. Biophys. Chem. 16 (1987) 583). The cooperative free energy value of the singly ligated species was approximately one-half the total cooperative energy. The cooperative free energy value of the doubly ligated species was not significantly different from that of carboxyhemoglobin. Because of experimental error, the observed difference in concentrations among the populations of the doubly ligated species cannot be taken as indicative of their functional heterogeneity. Additional studies on some NO intermediates have emphasized that (alpha 1 beta 1)(alpha 2 beta 2)X, a key intermediate in the formulation of the 'third-state' hypothesis in the deoxy/cyanomethemoglobin system, has a free energy value for dimer-tetramer assembly which is critically dependent on the nature of the ligand X as suggested by Ackers and Smith (reference as cited above).

Human globin chain separation by isoelectric focusing.

Human globin chain separation, a key procedure in the study of hemoglobinopathies, is routinely performed by chromatography on carboxymethylcellulose. This method, though relatively easy and highly reliable, is expensive and time consuming. A new procedure, based on isoelectric focusing, is presented which allows the simultaneous separation of globin chains from multiple samples (at least 20 per gel slab). The method is rapid, inexpensive and can be easily carried out in clinical laboratories, and its high sensitivity allows the identification of radioactive bands even with minute amounts of labelled material. A new phenomenon, called the 'Nonidet P-40 effect', which greatly enhances the separation between gamma and beta chains by binding to these two chains and shifting their pI values in opposite directions, is described.

Ctla4 and multiple sclerosis in the Italian population.

CTLA4 protein is a receptor molecule that plays a critical role as a negative regulator of the immune response. Therefore, genetic variations in CTLA4 may confer susceptibility to autoimmune diseases such as multiple sclerosis (MS). In order to investigate the association of two CTLA4 polymorphisms (+49 A/G and -318 C/T) with multiple sclerosis, sporadic MS patients and healthy controls from Italy were genotyped through direct DNA sequencing. Considering single-loci variations, no differences in the allelic and genotypic frequencies between patients and controls were found. However, considering a putative interaction at the two loci, the T/G combination was more frequently observed in patients than in controls. This result suggests that this allelic combination of the CTLA4 polymorphisms may be involved in the susceptibility to MS in the Italian population.

The specific influence of carbon dioxide and carbamate compounds on the buffer power and Bohr effects in human haemoglobin solutions.

1. The difference of pH (DeltapH) between human deoxygenated haemoglobin (Hb) and oxygenated haemoglobin (O(2)Hb) solutions when equilibrated with physiological pressures of carbon dioxide is (experimentally) much less than previously supposed.2. This smaller DeltapH is in contradiction to Wyman's (1948) theoretical calculations, wherein no allowance was made for the specific effect of carbamino-compounds on the amount of base neutralized by haemoglobin. Other previous authorities have also neglected this factor, which when properly allowed for restores the role of carbamino-compounds in CO(2) transport practically to that previously estimated by Ferguson & Roughton (1934a, b).3. At a given pH and P(CO(2) ), more CO(2) is bound by Hb solutions than by O(2)Hb, the difference increasing with pH. This result provides further, and seemingly decisive, evidence that the bound-CO(2) in the blood other than HCO(3)- (i.e. x-bound CO(2)) is oxygen-linked.4. According to a modified form of the Henderson-Hasselbalch equation for haemoglobin solutions [Formula: see text]. The value of pK(1)' is the same in Hb as in O(2)Hb solution and from the data in 3 is found to have the value 6.15 at 37 degrees C.5. The difference between the titration curves of O(2)Hb and Hb (DeltaX), at a given pH had been hitherto supposed to be the same in presence of CO(2) as in its absence. Our experiments show, however, that DeltaX is less in presence of CO(2) and at pH > 7.5 may even change sign. This paradoxical effect is also explicable, at any rate semi-quantitatively, by the effect of carbamino compounds on the buffer power, according to the theory put forward in the paper.6. The results show that the buffer power (dB/dpH) of haemoglobin solution under physiological conditions is 20-30% greater than previously estimated, and this also is in line with the new theory.7. In graphs of total CO(2) versus P(CO(2) ) in haemoglobin solutions (or blood) it has been customary to suppose that points on straight lines radiating from the origin are points of equal pH. Our data, however, show that the iso-pH lines drawn through the experimental points in the pH range, 7.2-7.4 do not, when produced as straight lines, pass through the origin, but intercept the P(CO(2) ) axis significantly to the right thereof.8. Calculations indicate that most of the x-bound CO(2) in haemoglobin solutions at pH 7.2-7.4 and at 37 degrees C can be accounted for by carbaminobound CO(2) without the need of postulating the existence of appreciable amounts of yet other forms, i.e. y-bound CO(2), in this range.

The binding of carbon dioxide by horse haemoglobin.

1. Three modified horse haemoglobins have been prepared: (i) alpha(c) (2)beta(c) (2), in which both the alpha-amino groups of the alpha- and beta-chains have reacted with cyanate, (ii) alpha(c) (2)beta(2), in which the alpha-amino groups of the alpha-chains have reacted with cyanate, and (iii) alpha(2)beta(c) (2), in which the two alpha-amino groups of the beta-chain have reacted with cyanate. 2. The values of n (the Hill constant) for alpha(c) (2)beta(c) (2), alpha(2)beta(c) (2) and alpha(c) (2)beta(2) were (respectively) 2.5, 2.0 and 2.6, indicating the presence of co-operative interactions between the haem groups for all derivatives. 3. In the alkaline pH range (about pH8.0) all the derivatives show the same charge as normal haemoglobin whereas in the acid pH range (about pH6.0) alpha(c) (2)beta(c) (2) differs by four protonic charges and alpha(c) (2)beta(2), alpha(2)beta(c) (2) by two protonic charges from normal haemoglobin, indicating that the expected number of ionizing groups have been removed. 4. alpha(c) (2)beta(2) and alpha(c) (2)beta(c) (2) show a 25% decrease in the alkaline Bohr effect, in contrast with alpha(2)beta(c) (2), which has the same Bohr effect as normal haemoglobin. 5. The deoxy form of alpha(c) (2)beta(c) (2) does not bind more CO(2) than the oxy form of alpha(c) (2)beta(c) (2), whereas alpha(c) (2)beta(2) and alpha(2)beta(c) (2) show intermediate binding. 6. The results reported confirm the hypothesis that, under physiological conditions, haemoglobin binds CO(2) through the four terminal alpha-amino groups and that the two terminal alpha-amino groups of alpha-chains are involved in the Bohr effect.

The association reaction between hemoglobin and carbon monoxide as studied by the isolation of the intermediates. Implications on the mechanism of cooperativity.

The concentrations of the intermediates in the association reaction between human hemoglobin and CO at 20 degrees C, pH 7, under conditions of negligible dissociation of the ligand, were measured by cryogenic techniques. The monoligated species were predominant at all values of overall ligand bound studied. The analysis of the experimental data assuming a scheme of four consecutive reactions indicated that the binding rates increased in a continuous fashion. A significant acceleration after the binding of the second molecule of ligand occurred in the presence of 0.1 M KCl, but not with the addition of an excess of inositol hexaphosphate, indicating that major functional, and possibly structural, transitions occur at the diligated state. Differences in the concentrations of the intermediates in the same state of ligation were observed under all conditions. The analyses of the data on the basis of schemes of multiple pathways of reaction indicated that the beta subunits reacted about 1.5 times faster than the alpha subunits in the first ligation reaction. After the addition of inositol hexaphosphate, the alpha subunits reacted about 1.5 times faster than the beta subunits in the first ligation step, but the overall rate of the first CO binding step was unchanged.

Hybrid formation for liganded hemoglobins A and C at subzero temperatures.

The kinetics of formation of the asymmetric carbonmonoxyhemoglobin hybrid (alpha beta)A(alpha beta)C from the parent molecules alpha 2 beta 2A and alpha 2 beta 2C have been studied by electrophoresis at subzero temperatures (down to -40 degrees C) using as supporting media gels of acrylamide/methylacrylate in dimethyl sulfoxide/water mixtures. It has been found that in these media the rate of hybrid formation is markedly affected by pH and decreases by an order of magnitude between pH 7.3 and 8.3. At pH greater than 10, t = -40 degrees C, the hybrid between alpha 2 beta 2A and alpha 2 beta 2C is stable for several hours. A rapid thermal quenching of a mixture of alpha 2 beta 2A and alpha 2 beta 2C prevented hybrid formation during the time required to separate the 2 molecules.

Isolation of intermediate valence hybrids between ferrous and methemoglobin at subzero temperatures.

Quenching a hemoglobin solution partially saturated with carbon monoxide into a hydro-organic solvent containing ferricyanide will produce under suitable conditions a population of partially oxidized and CO-bound hemoglobin molecules. Since each Fe3+ heme carries one extra charge, it should be possible, in theory, to resolve the spectrum of intermediate compounds between hemoglobin and carbon monoxide, which was originally present in solution. In this study we report: 1) the development of a simple and rapid method to quench aqueous hemoglobin solutions into a hydro-organic solvent at subzero temperatures; 2) the determination of suitable experimental conditions to isolate valence hybrids between carbonmonoxy- and methemoglobin by isoelectric focusing at temperatures as low as -25 degrees C; and 3) the identification and isolation of all valence hybrids of different charge between carbonmonoxy- and methemoglobin.

Effect of temperature on the p50 value for human blood.

We investigated the effect of temperature (19, 30, 37, and 43 degrees C) on the p50 value for normal human blood at pco2 = 5.72 kPa (43 mmHg), at various pHs (range 7.0 to 7.6) and molar ratios of [2,3-diphosphoglycerate]/[Hb4] (range 0.4 to 2.4). The d(log p50)/d(pH) coefficient varied from 0.39 at 19 degrees C to 0.35 at 43 degrees C. The relationship between log p50 and 1/T (T = degrees Kelvin) was linear under the experimental conditions used, and the d(log p50)/d(1/T) coefficient varied between -2138 at pH 7.0 and -2162 at pH 7.6, independent of the concentration of 2,3-diphosphoglycerate. Assuming that the effect of pco2 on the p50 value is the same at 19, 30, and 43 degrees C as at 37 degrees C, one can use the reported coefficients to calculate the p50 value for normal human blood under conditions of temperature, pH, pco2, and 2,3-diphosphoglycerate concentrations prevailing under physiological and pathological conditions. The p50 value calculated by empirical equations, taking into account the effect of temperature, correlated well with the values for p50 determined experimentally (y = 0.9774x + 0.453; r = 0.998; n = 60), with an SD of 52 Pa (0.39 mmHg).