312 



BIOCHEMICAL GENETICS 



the solubility of the salt must be sufficient to allow a precipitating concentration to be 

 reached; and the pH of the buffer should be near the isoelectric point of the protein, 

 at which point solubility is generally minimal. The most commonly used buffer 

 for hemoglobin studies is potassium phosphate. The hemoglobin is usually converted 

 to carbonmonoxyhemoglobin, since the latter is more stable than oxyhemoglobin. 

 The amount of carbonmonoxyhemoglobin left in solution is determined photometrically. 

 To compensate for daily variations in the physical conditions, standards are usually 

 run along with each group of assays. Popp 1014 has modified this technique by establish- 

 ment of nomographs to enable quantitative determinations of mixtures of several 

 murine hemoglobins within + 5 per cent. 



Fig. 43. Comparison of murine carbonmonoxyhemoglobins analyzed by 



CRYSTALLOGRAPHIC METHODS. 



A B 



Precipitates were formed within 21 hours; 3.08 M phosphate buffer, pH 6.5, 24° C. 



Left (A). Crystals of single hemoglobin of strain C57BL mice. 



Right (B). Amorphous precipitate of diffuse hemoglobin of strain 101 mice. 



Literature on the solubility of hemoglobin is not extensive 65 - 66> 478 ' 670 ' 687 ' 1068 

 in view of the fact that a solubility study may be performed with little effort and ex- 

 pense in conjunction with other types of assay. A relationship between electrophoretic 

 and solubility characteristics may exist, as was found for the sheep 1338 and mouse. 1015 

 Solubility may also be helpful in distinguishing hemoglobin types that are not electro- 

 phoretically different. Itano 645 showed that although human hemoglobins D and S 

 are electrophoretically similar, the reduced form of hemoglobin D is more soluble 

 in phosphate buffer than the reduced form of hemoglobin S. More recent studies 

 have indicated other differences between these hemoglobins which will be discussed 

 in the next section. In studies on the inheritance of single and diffuse hemoglobins 

 among inbred strains of mice, Popp (unpublished data) has observed that several 



