Arda Alden Green 223 



taminant is shown by the difficulty of removing this bound carbohydrate. 

 Prolonged fractionation with salts, isoelectric precipitation, dialysis, heat 

 coagulation and even specific flocculation between antigen and antibody all 

 failed to remove the carbohydrate present in serum proteins" (p. 1502). 



If one studies combination with any one substance, the proteins may be 

 relatively easily separated into groups having a constant percentage of that 

 constituent. If more than one compound is considered the problem is much 

 more difficult. For instance, the number of euglobulins of serum are multi- 

 plied if one considers both phospholipid and carbohydrate content. Un- 

 doubtedly, there are also other substances combining with proteins. 



Proteins combine not only with other substances but with each other. The 

 problem of the identification of the euglobvdins is further complicated by the 

 ability of water-soluble globulins to unite and form water-insoluble globulins 

 behaving like Pj. This was first recognized by Kendall,^* working on human 

 serum. He noted a precipitate formed on the addition of the less soluble 

 pseudoglobulin to the more soluble pseudoglobulin. He showed quantitative 

 relationships depending upon pH, analyzing the protein content by im- 

 munological means. Hewitt" has since shown that his pseudoglobulin A from 

 human serum will form a precipitate with the globoglycoid separated from 

 the serum of different species. It is evident that pH is especially important 

 in trying to separate these two proteins. 



Ever since S0rensen's* classical studies on the solubility of crystalline serum 

 albumin it has been known that there must be more than one serum albumin, 

 since solubility was not independent of the amount of solid crystals present. 

 Two albumins have now been crystallized from horse serum having different 

 electric mobility and different carbohydrate content. One is carbohydrate 

 free and one contains 5.5 per cent of carbohydrate.^" ^"^ The carbohydrate-free 

 protein may also be crystallized from concentrated salt-free solutions as the 

 sulfate. "° Although successful studies on the solubility of fibrinogen had been 

 carried out previously/^ the carbohydrate-free albumin is the only protein so 

 far isolated from serum exhibiting constant solubility independent of the 

 amount of saturating body. Carbohydrate-free crystalline human serum albu- 

 min shows the same evidence of purity.^ 



Other proteins so far found in the albumin fraction are enzymes, including 

 choline esterase and phosphatase, a crystalline copper containing protein, 

 hemocuprein,^' seromucoid,"' and a certain amount of euglobulin. 



The solubility curves of these serum proteins necessarily overlap tremen- 

 dously. Advantage must be taken of all possible variations in (i and K^. That is, 

 differences in variation of solubility with temperature, with pH, with kind 

 and concentration of electrolyte, and with type of solvent must all be utilized. 

 Separation can be effected only after repeated fractionation, the precipitating 

 agent being added in small increments under controlled conditions, including 

 control of the protein concentration, and careful analysis of each fraction be- 

 fore pooling with other similar fractions. 



