Simon H. de Bruin

The interaction of chloride ions with human hemoglobin

Abstract Studying the effect of KCl on the Bohr effect of human hemoglobin, it appeared that at low Cl− concentration the alkaline Bohr effect is considerably smaller than it is at a Cl− ion concentration near 0.1 M. The data show that at least part of the Bohr effect, that thus far could not be attributed to a particular residue in hemoglobin, is due to interaction of hemoglobin with anions. The effect of KCl on the Bohr effect shows a striking similarity with the effect of 2,3-diphosphoglycerate (DPG) on the Bohr effect. Based on this a mechanism is proposed which satisfactorily explains the observed salt effect.

The kinetics of carbon monoxide binding to partially reduced methemoglobin

Abstract The pulse radiolysis technique has been used to study the kinetics of the CO binding to partially reduced methemoglobin. Experiments with horse heart metmyoglobin show that this technique gives results which are in good agreement with those obtained by other methods. The kinetics of the CO binding to partially reduced methemoglobin show two phases, whose amplitudes appear to depend on the degree of reduction in such a way that they can be attributed to methemoglobin molecules with one or two reduced heme groups. In the presence of inositol hexaphosphate the rate of CO binding to partially reduced methemoglobin decreases strongly. With inositol hexaphosphate a slight biphasic behavior is observed independent of the degree of reduction.

Pulse-radiolytic studies on the spin-state transitions in aquomethemoglobin after reduction of a single heme group

We have previously observed a transient state (halftime ‘L 15 ps) in aquomethemoglobin with an absorption maximum near 420 nm after rapid reduction of a single ferric heme group by hydrated electrons [l] . This observation has been confirmed [2,3] . Moreover, this microsecond process disappeared in the presence of Ins-P6 [l] . On binding this organic phosphate, metHb changes its quaternary conformation from the Rto the T-state [4]. Therefore, we concluded that in the absence of an allosteric effector the reduction reaction of one heme group in metHb by hydrated electrons was followed by a quaternary conformational change (R + T transition). spin-state. The subsequent transition of the ferrous low spin to the stable ferrous high spin-state proceeds with a half-time that depends on the solvent condition. On the basis of these results we have postulated a two-step reduction mechanism for aquomethemoglobin.

The influence of organic phosphates on the bohr effect of human hemoglobin valency hybrids

The Bohr effect of hemoglobin and that of the aquomet and cyanomet valency hybrids was measured in the presence and the absence of IHP (inositol hexaphosphate) and DPG (2,3-diphosphoglycerate). In the absence of these organic phosphates the four hybrids show similar, but suppressed Bohr effects as compared to hemoglobin. Addition of IHP and DPG results in all cases in an increase of the Bohr effect. The additional phosphate induced Bohr effect of the hybrids with the alpha chain in the oxidized form is almost identical to that of hemoglobin, while this effect of the hybrids with oxidized beta chains is slighly lower than that of hemoglobin. The results suggest (a) that the Bohr effect is correlated to the ligation state of the hemoglobin molecule rather than to its quaternary structure (b) that the additional phosphate induced Bohr effect is related to the change in quaternary structure of the tetramer, and (c) that with respect to the Bohr effect of the hybrids there is no difference between high and low spin species.

Differential hydrogen ion titrations of the histidine residues in Helix pomatia haemocyanin

The properties of the histidine residues in Helix pomatia haemocyanin have been studied by differential hydrogen ion titrations. In oxy-and deoxyhaemocyanin 31 X 10(-5) histidine residues per g protein are titrated in contrast to 35 X 10(-5) residues in apohaemocyanin. The difference corresponds to a stoichiometry of one histidine residue per copper atom bound. Even in apohaemocyanin about 6 X 10(-5) histidine residues per g protein are not titrated in their normal pH region. In the presence of sufficient calcium to displace the dissociation completely out of the titration region, the titration curve of apohaemocyanin could be linarized according to the model of Linderstrom--Lang. In oxy-and deoxyhaemocyanin, however, a distinct deviation from linearity was found under the same conditions. In the absence of calcium the effect of the dissociation adds up to this deviation. The electrostatic interaction factors were determined for the protein at 0.1 M KC1 and for the dissociation products: halves and tenths at 1.0 M KC1. The electrostatic interaction factor for the wholes and the halves are much smaller than the values calculated from the Linderstrom--Lang equation, using the radius of the equivalent sphere either obtained from electron microscopy or from the partial specific volume. This probably due to solvent penetration. For the tenths at 1.0 M KC1, this effect is small.

The CO and NO bohr effect of human hemoglobin with and without inositolhexaphosphate

Using NO and CO as ligands the Bohr effect of human hemoglobin has been measured with and without inositolhexophosphate. It appears that in the absence and presence of inositolhexaphosphate hemoglobin shows a distinct ligand specificity with respect to the Bohr effect. Ligation with NO is accompanied by release of a larger number of Bohr effect. It is shown that this latter result is due to the fact that the number of protons taken up upon binding of inositolhexaphosphate to ligated hemoglobin is larger for HbNO than for HbCO. It is suggested that this additional proton uptake is partially due to a restoration of the saltbridge between His 146beta and Asp 94beta upon addition of IHP.

Some aspects of cooperativity in human hemoglobin

Abstract The cooperativity in hemoglobin can be described by the Hill parameter n , the free energy of interaction Δ F 1 and the allosteric free energy Δ F A . By this latter is meant here the free energy change associated with the transition from the deoxy to the oxy conformation in hemoglobin. In this paper some general relations between n , Δ F 1 and Δ F A are given. A method is presented by which Δ F A can be calculated from oxygenation data.