Serum Protein Fractionation

Only two methods of separating serum protein components are sufficiently simple and convenient to be useful in routine work. Salt fractionation is easy to carry out, involves no special equipment, and, if done carefully, provides very useful clinical information. Commonly, only separation of albumin from the globulins as a group is carried out, but techniques are available for separating and quantitating the chief globulin components, especially gamma globulin. When more precise values of the protein fractions are desired, electrophoretic techniques are used. These require more skill and the use of special equipment, and they are more time consuming, although with cellulose acetate membranes, separations may be made in less than an hour. Ultracentrifugation, requiring an expensive instrument, is not suitable for routine use, but is especially useful in the study of lipoproteins and macroglobulins.

Salt Fractionation; Albumin-Globulin Ratio
Albumins are soluble in water, whereas globulins are not. If an electrolyte solution of low concentration (0.1 M) is added to a globulin, the latter will go into solution by virtue of the phenomenon of salting-in. The cations and anions of the electrolyte bind to reactive groups on the protein molecules and break the bonds holding the protein micelles together, allowing the individual protein molecules to undergo solution. The salt concentration of plasma, approximately 0.15 M, is ideally suited to maintain globulins in solution. As the salt concentration is increased to high levels (of the order of 2 M), the various globulins will again become insoluble and will precipitate by salting-out. The level of electrolyte needed to precipitate the various globulins will vary with the individual protein and electrolyte and will also depend on pH and temperature.

Ammonium sulfate was the first salt to be used and it is still used in protein and enzyme investigations. Those proteins precipitating in one-third saturated ammonium sulfate solution were termed euglobulins (true globulins). As the salt concentration is increased, the pseudoglobulins begin to precipitate out, and at concentrations near 50 per cent saturation, all globulins become insoluble, the proteins still in solution constituting the albumins. The use of ammonium sulfate for the isolation and assay of gamma globulins will be described later. The salt is not used in routine clinical work because it interferes with the biuret reaction and obviously cannot be used with the Kjeldahl nitrogen technique.

The only salts whose use has become established in routine clinical chemistry are sodium sulfate and sodium sulfite. The former (Na2SO4) was used as early as 1901, but its large scale use followed the work of Howe in 1921, who recommended the use of 22 per cent (w/v) sodium sulfate to precipitate globulins. Total protein was assayed, and, after removing globulins by filtration, the albumin fraction was measured in the filtrate, globulin being calculated by difference. In 1940 Kingsley introduced the use of ether as a means of separating the precipitated globulins from the soluble albumins, without resorting to the slow, tedious filtration. By carefully dispersing diethyl ether into the mixture of serum plus salt solution, the insoluble globulin micelles would entrap sufficient ether so that on centrifugation, instead of sedimenting as they would normally, the globulins would float above the albumin solution as a compact pellicle. With this type of procedure, serum albumin was found to range from 4.0 to 5.5 gm./100 ml., the globulin from 2.0 to 2.9 gm./100 ml., and the albumin/globulin ratio from 1.5 to 2.5 (average, 2.0).

With the development of Tiselius' moving boundary electrophoresis technique, it was logical to compare the electrophoretic albumin fraction with that separated by the use of Howe's 22 per cent sodium sulfate. It was immediately evident that the Howe technique yielded higher values because it included with the albumins, the alpha-1 and some of the alpha-2 globulins. Studies by Majoor and Milne showed that to free the albumin of all globulin fractions, the concentration of sodium sulfate must be of the order of 26 to 27 per cent (w/v) (1.9 M). Unfortunately, this concentration is beyond the solubility of sodium sulfate at room temperature (25 degree of Celcius), and the salt solution must be kept at 35 to 37 degree of Celcius to avoid crystallization of the salt on storage and during use. With the use of 26 per cent sodium sulfate, the albumin/globulin ratio is of the order of 1.1 to 1.8, averaging about 1.4; this agrees quite well with that obtained by electrophoretic methods. Other salts have also been employed. Campbell and Hanna used sodium sulfite, obtaining complete separation of globulins from albumin at 27 per cent (w/v) (2.2 M). This salt has the advantage of being more soluble than sodium sulfate, and, because it acts as a buffer, better pH control of the precipitation reaction is possible. Reinhold proposed the use of a mixture of sodium sulfate plus sodium sulfite and this procedure will be presented in detail.

By using varying concentrations of sodium sulfate (or sodium sulfite), it is possible to separate all of the main globulin fractions from one another. For example, Kibrick and Blonstein used 15 per cent (w/v) sodium sulfate to precipitate the gamma globulins and 26 per cent salt concentration to precipitate all globulins. The difference between these values gave the sum of alpha and beta globulins. By further use of 19 per cent sodium sulfate, which precipitated the beta- plus gamma globulins, the amount of alpha globulins could also be determined. Such fractionations are tedious, however, and not too satisfactory, especially when abnormal sera are used, and they have never become popular. The electrophoretic separation is simpler and considerably more accurate.