Paper Electrophoresis of Serum Protein in the Acid Buffer with Special Reference to the Electrophoresis at pH 4.4 of Mc Ilvaine's Buffer

Abstract

Paper electrophoretic studies of serum protein iu the acidic Mc Ilvaine's buffer (pH 2.4 to 6.0) were carried out, using the open horizontal method. Further, the reproducible paper electrophoresis technique at pH 4.4 and the subsequent quantitative methods were reported.
The following results were obtained.
1. The acidic protein in serum, named M, is separated only between pH 4.8 and 4.0. At pH 4.8 and above, serum protein is separated into three fractions, namely, A, B₁ and B₂; no acidic protein is observed. At pH 3.6 and beneath, , serum protein is not separated in any fraction, and distributed only as a long and diffuse tailing on the cathodic part of paper.
2. Serum fractions at pH 4.4 are named M₁, M₂, A and B from the anodic components. B, having no electric charge at pH 4.4, is displaced only by the buffer flow. M₁, M₂ and A migrate to the anode with the apparent mobilities in this order, but the migration of A is very small. B alone migrates to the cathode.
3. Under the constant voltage of 200V, the electrophoretic separation at pH 4.4 is markedly influenced by the ionic strength of buffer solution and electrophoresis hour, namely, 1) with lower ionic strength, M₁, M₂, A and B are so far well resolved but their zones are diffuse and blurred, and 2) with much higher ionic strength, and also in too longer electrophoresis hour, they come together on the central part of paper because the buffer flow and the ionic strength on paper are exceedingly large.
4. Serum should be applied at the places where A remains immobile after electrophoresis. It is because B fuses with A, when the applied volumes of serum are relatively large, and because the fast moving M₁ and M₂ are flowed back owing to the buffer displacement and superimposed on the other fractions, if the application points are on the more anodic part than the above mentioned places.
5. In the author's laboratory, electrophoretic runs are routinely carried out at 200V for 14-16 hours, using Mc Ilvaine's buffer of pH 4.4 and 0.2 which contains 3.29g of citric acid (1 hydrate) and 10.53g of sodium diphosphate (12 hydrates) in a litre of destilled water.
6. By amido black staining, M₁ is stained only faintly, M₂ weakly, A and B exceedingly densely. By PAS-staining, M₁ and M₂ are densely stained only in cases with increased mucoproteins. A and B, in general, are faint and diffuse. Therefore. these staining methods are inadequate for the quantitative densitometry of these fractions.
7. Sera from some representative dysproteinemias including liver cirrhosis, gastric cancer, acute myelocytic leucemia, nephrotic syndrome and exudative pleuritis were run at pH 4.4 and fractionated. Subsequently, serum fractions and their protein-bound hexoses were chemically determined and compar ed.
8. The improved preparative electrophoresis by paper strips (Yamaguchi) is proved to be valuable for the clinical observation of serum pH 4.4 as well as for the further investigation of M₁ and M₂ fractions.