The Determination of the Velocity Constants of the Four Successive Reactions of Carbon Monoxide with Sheep Haemoglobin

It is now generally accepted that the combination of carbon monoxide (or oxygen) with reduced mammalian haemoglobin takes place in four stages expressible in terms of the four intermediate reactions Hb4+ CO → Hb4(CO) (combination velocity constant l΄1), Hb4(CO)+CO Hb4(CO)2(l΄2),Hb4(CO)2+CO → Hb4(CO)3 (l΄3), and Hb4(CO)3+CO → Hb4(CO)4(l΄4), as follows from Adair’s original suggestion thirty years ago. Hitherto, experimental data on the overall rate of combination of carbon monoxide with sheep haemoglobin have not been precise enough to permit the direct determination of the individual velocity constants to l΄1 to l΄4. Recently, however, helped by the evaluation of l'4 from equilibrium measurements by Roughton, and using the stopped flow technique for following the combination reaction, it has been possible with the aid of automatic electronic computation and modem statistical methods, to evaluate the three remaining velocity constants, together with their standard errors. The corresponding coefficients of variation of the estimated values of the velocity constants lie within the range of 4 to 12%. Data have been obtained under standard conditions (pH 9∙1 and 20° C) as well as at the physiological pH of 7∙1. The effect of temperature has also been studied. If there were no haem-haem interactions the four constants l΄1 l΄4 to would stand in the relation 4:3:2:1. Under standard conditions, we find 4:6:2:80, and at pH 7∙1, 4:3:12:80 for sheep haemoglobin. It is clear that a striking change in the mode of combination takes place after three molecules of CO have already combined, a finding confirming that a marked mternal change in the molecule then takes place. The fact that l΄3 and l΄ 3 are unequally affected by a change of pH from 9∙1 to 7∙1 constitutes a further exception to the principle that change of pH (or of temperature) only affects the scale but not the shape of the equilibrium curve and the velocity curves for the reactions of haemoglobin with oxygen and carbon monoxide.