J. H. P. JONXIS 



chromogen. At a wavelength of 650 mu there is a considerable 

 increase in the light absorption during this denaturation {Figure 1), 

 and this increase can be used for the determination of the amount of 

 oxyhaemoglobin which has not yet been denatured. By measuring 

 at intervals of one minute the extinction of a 0-2 per cent solution 

 of haemoglobin to which has been added buffer of pH 12-7 it is 

 possible to calculate the percentage of oxyhaemoglobin still present 

 at different times. The values found in this way can be used 

 to make a curve (Figure 2). On the horizontal axis the time is plotted 

 in minutes, and on the vertical axis the logarithm of the percentage 

 of oxyhaemoglobin still present. If there is only one haemoglobin 

 present in the solution the values will form a straight line ; this means 

 that the denaturation behaves as a monomolecular reaction. When 

 there are two different forms of haemoglobin in the solution with 

 different resistances to alkali the values will form two straight lines. 

 By extrapolating back to the vertical axis the line which is formed by 

 the values of that fraction of haemoglobin which denatures more 

 slowly, it is possible to calculate the percentage of this more resistant 

 haemoglobin. In human beings the foetal haemoglobin is far more 

 resistant than the adult one. The rate of denaturation depends on 

 the kind of haemoglobin, the pH, the concentration of the buffer 

 solution and the temperature. When buffer solution is used with a 

 pH of 12-7 the denaturation of later haemoglobin is rapid while that 

 of foetal haemoglobin is slow. Thus it is possible to calculate exactly 

 the amounts of foetal haemoglobin present in a mixture of the two 

 forms 5 ' 6 . 



20 



Figure 2. Rate of alkali-denaturation of a 

 02 per cent solution in 1 N NaOH of foetal 

 Hb, later Hb and of a mixture containing 

 25 per cent of foetal Hb. Temperature 20°C 

 Vertical axis : log. percentage unchanged Hb. 



2 4 6 8 

 Time in Minutes 



Figure 3. Rate of alkali-denaturation at pH 

 11*7 and 25 °C of haemoglobin from an adult 

 man. Haemoglobin concentration 0-2 per cent. 



20 



16 



c* 12 



08 



'04 



4 8 12 16 



Time in Minutes 



262 



