Observations on foetal haemoglobin.

Foetal haemoglobin exhibits a much greater resistance towards alkaline denaturation than does adult haemoglobin, thus constituting an important distinction between the two types of haemoglobin. The phenomenon was first described by Korber (1866), observed again by Kruger (1887-88), and subsequently studied in more detail by Bischoff (1926), Haurowitz (1929, 1930), and Brinkman, Wildschut and Wittermans (1934). The latter developed a photoelectric method that enabled them to work with mixtures of pigments having different degrees of resistance to alkaline denaturation. Further study has demonstrated that the two types of haemoglobin differ also in many other properties (Table 1). However, the most pronounced difference so far described is to be noted in the speeds of denaturation by alkali, but the mechanism is not yet understood. No report could be found in the literature describing the behaviour of the two types of haemoglobin under the influence of other denaturing agents. Among such agents, urea and salicylates are known to bring about denaturation of haemoglobin, and at the same time maintain the thus denatured protein in solution. Urea in concentrated solution causes denaturation of haemoglobin and, in addition, horse (Astbury and Lomax, 1935) or ox (Huang and Wu, 1930) haemoglobin molecules are dissociated into half molecules as determined by osmotic pressure measurements. The effect ofvarying concentrations of urea and related amides on horse haemoglobin has been studied extensively by Steinhardt (1938) who found, when working with horse methaemoglobin formed by the action of potassium ferricyanide, that within 20 hours at room temperature the characteristic absorption spectral band in the red (630(h) fades and is replaced by a diffuse band in the green (at approximately 534,4). It was concluded that the latter band was not due to ferrohaemochromogen since, on treatment with sodium hydrosulphite, the typical absorption spectrum of ferrohaemochromogen did not appear. The denaturing effect of sodium salicylate on bovine methaemoglobin was examined in detail by Anson and Mirsky (1934). They neasured the increase in the spectral absorption in the green and found that when salicylate was added to native methaemoglobin the absorption in the green at first increased rapidly, then remained constant and finally increased again very slowly. The concentration of salicylate in their experiments varied from 0-05 M to 0-5 M and they observed that greater proportions of denatured protein were produced and with increasing concentration of salicylate the speed of reaction was also increased. Using 0-5 M salicylate solutions denaturation was complete in 30 minutes. In the present study we have subjected foetal and adult methaemoglobin to the denaturing action of urea and salicylate and studied the behaviour of the two pigments. These two denaturing agents were chosen since, in the concentrations used, they possess the property of keeping the denatured protein in solution, thus facilitating spectroscopic studies and photometric determinations.