278 VI. HEMOGLOBIN 



the dissociation curve and the redox potential of the mode of prepara- 

 tion and the species of animal used. 



6. KINETICS OF HEMOGLOBIN REACTIONS 



6.1. Methods 



Not until Hartridge and Roughton commenced their work in 1923 

 did it become possible to approach the hemoglobin equilibria from 

 the standpoint of their kinetics. The velocities of the reactions they 

 set out to measure were extremely fast, and the original papers 

 should be consulted to get a proper idea of the ingenuity with which 

 they surmounted the technical difficulties. In principle, their method 

 consisted of observing the spectrum of the compound in which they 

 were interested, at various positions along a tube through which the 

 hemoglobin solution was driven at a high velocity, a few milliseconds 

 after it had been effectively mixed with an aqueous solution of the 

 substance with which it was reacting. Their analyses were based on 

 measurement of the position of the a band of a mixture of carboxy- 

 hemoglobin and oxyhemoglobin with a Hartridge reversion spectro- 

 scope. 



The early technique for measuring the kinetics of these fast 

 reactions was subsequently altered by Roughton and Millikan {2368) 

 by the replacement of the Hartridge reversion spectroscope by a 

 differential photoelectric cell arrangement, when, by the use of proper 

 wavelengths of light, a potential difference could be established 

 which was proportional to the saturation of the pigment. While the 

 earlier techniques required liters of blood, this improvement enabled 

 a reduction to be made in the scale of the apparatus, and in Millikan's 

 modification {1952a) the solutions are delivered by motor-driven 

 syringes and a complete investigation of the kinetics of the pigment 

 can be made with a few milliliters of solution. 



The work on the kinetics of the hemoglobin and myohemoglobin 

 systems was carried out during the period 1923 to 1936. This period 

 was one in which great advances were made in the study of hemo- 

 globin, some of which necessitated reinterpretation of the early 

 kinetic investigations. In this chapter, we are concerned more 

 with the mechanism of the reactions than with the absolute values 

 obtained. 



