The kinetics of hæmoglobin.—II. The velocity with which oxygen dissociates from its combination with hæmoglobin

Abstract
The velocity with which oxygen combines with, and is dissociated from, hæmoglobin is a matter of considerable interest both to the physiologist and the physical chemist; to the former because of the all-important part performed by this compound in respiration, and to the latter because hæmoglobin is an almost unique example of a large complex protein molecule which combines with gases, apparently not by adsorption, but in a simple chemical manner defined by the laws of mass action. There were several preliminary problems which it was necessary for us to solve, before our main experimental investigation could be commenced. Our first major problem was to find some very sudden method of upsetting the chemical equilibrium subsisting between oxygen, hæmoglobin and oxyhæmoglobin in solution. The time taken to upset the equilibrium must be very much shorter than the time taken by the system to regain chemical equilibrium. This problem in the case of the reaction CO+O2Hb⇄O,+COHb was solved by exposing the solution to a powerful beam of light; the latter caused a new position of equilibrium to be taken up, and this could be instantaneously upset by a sudden interruption of the beam of light. The system thereupon returned to its position of dark equilibrium. In order that all parts of the solution shall be passing through the same stages of the resulting reaction it is necessary that the time taken for the equilibrium to be disturbed be of negligible duration compared with that taken for equilibrium to be regained. Unfortunately, a similar method was not open to us in the present case, for the reaction O2+ Hb ⇄ O2Hb is not appreciably, if at all, affected by a powerful beam of light. The factors upon which the equilibrium of this system depends have been very thoroughly studied by Barcroft and his co-workers in recent years (18) ; the principal ones are the temperature, the hydrogen ion concentration and salt content of the solution. Calculations showed that even if we had some very sudden method of changing one or more of these factors, the amount by which the system would be displaced would be too small compared with the experimental error of the quantitative methods available. The plan which we have therefore adopted was to prepare a hæmoglobin solution I and another solution II such that if I and II are very rapidly,but completely, mixed, the solution immediately after mixing is not in chemical equilibrium, but reaches equilibrium after an interval of time which is long in comparison with the time taken up by the process of mixing. Thus as an example we may mention that, in studying the rate of oxidation of hæmoglobin, solution I consisted of dilute reduced hæmoglobin, whilst solution II consisted of water containing sufficient dissolved O2to combine with the hæmoglobin of I. At the instant after the very rapid mixing of I and II the hæmoglobin is still partially reduced, and by methods to be described later the rate at which it subsequently becomes fully oxidised is measured. For this plan to be successful it was necessary to devise a special type of mixing apparatus, the description and testing of which have already been described in one of our previous papers (2).

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