Absorption of Haemoglobins Inside and Outside the Red Blood Cell 



on changing the pH from neutrality to the region of pH 9-10. On 

 standing for 24 hours at room temperature at such alkalinity the first 

 intermediate denaturation state noted above is observed 6 . In more 

 alkaline solutions the Hb0 2 becomes rapidly denatured (as methae- 

 moglobin), as it does also on the acid side of pH 5-5. The Soret 

 bands of HbCO 6 and of methaemoglobin 7 ' 8 remain entirely unchanged 

 both in wavelength and in intensity up to at least /?H 9, and the visible 

 bands of all these compounds are insensitive to pR changes so long 

 as the protein is not denatured, with the exception of the 635 mjx 

 band of methaemoglobin 9 ' 6 . which changes over the range pH 6-10. 



Figure 3. Spectral absorption 

 of human adult oxyhaemoglobin 

 in the Soret band region, show- 

 ing adherence to Beer's law 

 from 0.193 per cent to 38.8 per 

 cent oxyhaemoglobin. 



100 



J80 



■400 420 



A (m/j) 



440 



Within the red blood cell the concentration of the haemoglobin is 

 about 35 per cent, and in extending these spectral studies to intact 

 red cells the spectral absorption of haemoglobin at these high con- 

 centrations must be known. Drabkin and Austin 9 have shown that 

 in its visible spectral absorption Hb0 2 obeys Beer's law up to these 

 high concentrations, and I have been able to show that it obeys Beer's 

 law in the Soret band region also in concentrations from 0-019 per cent 

 to 38-8 per cent {Figure 3). The high molar extinction of this band 

 requires the use of very thin layers when concentrations as high as 



38-8 per cent are used : E 



(414-5 m^) 



for a 5\x layer of a 38-8 percent 



Hb0 2 solution is 1*5. Split mica spacers were used between quartz 

 plates to obtain these thin layers, and the large range for the limiting 

 curves of the spectral absorption of the strong solution {Figure 3) is 

 due to the uncertainty of measurement of the mica spacer thickness. 

 It must also be remembered that the refractive index changes at the 

 quartz/solution interfaces are somewhat different for the 0-019 per cent 



209 



H— 14 



