Separation of Albumin from Globulins

Toward the end of the nineteenth century, European pharmacologists demonstrated the heterogeneity of the protein (albumen) in blood serum. Concentrated salt solutions were employed to precipitate the globulins. After the work of Kander (K2) and Lewith (LIO) the serum protein, which remained in solution after mixing diluted serum with an equal volume of ammonium sulfate solution saturated at 25°C (final concentration 2.05 Af), was designated albumin.  

New interest in the nature of the constituent serum proteins resulted from the introduction of the sodium sulfate fractionation scheme by Howe (H24, H25). This was a technical advance because, unlike ammonium sulfate, sodium sulfate need not be removed prior to nitrogen analysis, the only accurate available means at that time of determining protein. Howe (H24) observed three ranges of sodium sulfate concentration at which an increase in molarity of 0.05 in a 1 30 dilution of calf serum failed to produce the expected increase in precipitation. One of these critical concentrations occurred at 1.45-1.50 M and was thought to indicate complete globulin precipitation. 

The concept of proteins as heterogeneous colloidal systems rather than as discrete molecules tended to discourage the development of new separation processes. Protein separation with other salts, e.g., magnesium sulfate and mixtures of the hydrogen phosphates of sodium and potassium, were investigated, but in the studies undertaken the salt concentrations in the protein mixtures rarely exceeded half-saturation. 

Although as many as eight distinct proteins have been demonstrated immunologically in the soluble fraction obtained at 65% ammonium sulfate saturation (2.6 M), the total amount of these antigens represents a very small part (about 3%) of the total albumin fraction (06). 

The time allowed by Howe for the precipitation of serum globulins in 1.5 M sodium sulfate (H24) was 12 hours. Most workers who have studied this separation procedure have used from 1 to 4 hours at 37 C (H17, K16, K19, L8, R16). Fine (F5) used a precipitation time of 15 minutes and more recently Rappaport and Loew (R4) found 5 minutes sufficient. 

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Separation of albumin from globulin, originally done by filtration, was greatly facilitated by Kingsley s introduction of the ether technique (K20). Ether is added to the salt-protein mixture, which is then repeatedly mixed by inversion for 20 seconds and subsequently centrifuged. Flotation of the globulins ensues, and the albumin fraction separates as a subnatant solution. 

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