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Isolated albumin almost always contains polymerized forms which appear during preparation and storage of the protein. The proportion of polymerized forms reflects the degree of stability of the solution. The quantitative estimation of the polymers is usually performed by gel chromatography. In this work, the high resolution power of polyacrylamide gradient gel electrophoresis (Gradient PAGE) was used to separate the polymers present in the preparations of human serum albumin. The analysis of the different peaks obtained by gel chromatography allows to conclude that peak 1 contains aggregates and high polymers, peak 2 trimer and dimer and peak 3 the monomer of albumin. The aggregates of the peak 1 can be dissociated by SDS and correspond, in gradient PAGE, to the high polymers. By using gradient PAGE in the presence of SDS under the conditions described in this paper, it is possible to estimate the proportion of high polymers and aggregates present in albumin preparations. These results are similar to those obtained by chromatography followed by a protein assay but noticeably inferior to those resulting from measurements performed by absorbance at 280 nm.

Inter-alpha-trypsin inhibitor (I alpha I) has been purified from C.N.T.S. fraction III as starting material. The purification procedure includes D.E.A.E. cellulose chromatography and gel filtration on G 150 Sephadex in the presence of EDTA. The purified protein gives one precipitation line in immunoelectrophoresis against anti-whole human sérum. It reacts only with an anti I alpha I immune serum and possesses a strong antitryptic activity. When studied in starch or polyacrylamide gel electrophoresis 2 components are observed, each of them having the same antigenic structure and the same antitryptic activity as the crude preparation. The slower and less important component is dissociated by 0,1% SDS. The molecular weight estimation of I alpha I BY PAA/SDS is about 180,000. This result is not modified by the presence of 1% beta mercaptoethanol indicating that I alpha I consists of one polypeptide chain. Crude preparation reveals under the same electrophoretical conditions small amounts of low molecular weight components (135,000 52,000 and 26,000) which can be due to a proteolytic action on I alpha I. Indeed plasmin is able to produce such fragments having an antitryptic activity as shown by fibrin/polyacrylamide gel electrophoresis. The relationship between small molecular weight inhibitors of human serum and bronchial secretions and those obtained after degradation of I alpha I by plasmin is discussed.

In order to clarify the actual nomenclature of the albumin allotypes in French and Italian populations we compared the samples collected in our laboratory to those kindly supplied by Dr. Porta (Italy) with reference to albumin variants classified in starch gel electrophoresis using three buffer systems by Weitkamp. The inherited human albumin variants can be classified on the basis of their relative mobilities on cellulose acetate electrophoresis compatively to human transferrin mobility. The relative mobility of each variant can be expressed by the following ratio: migration distance of the variant versus migration distance of the normal albumin where zero represents the transferrin mobility. Using three buffers system at pH 8.6, 5.0 and 6.9, it is possible to distinguish some albumin variants having a same mobility at alkaline pH and different mobilities at acidic pH. In the european area, eleven albumin variants are distinguishable on the basis of their relative mobilities at different pH: four Fast moving variants: Gent, Vanves (a new variant described here), Reading and CN/BL, and seven Slow moving variants: MI/MI Slow, GE/CT, SO/BS (or D type), Pollibauer, Gainesville, Roma and B type. Thirty-six sera from unrelated subjects with genetic bisalbuminemia were analyzed in our laboratory. Their distribution was as follows: B type (22), Pollibauer (9), SO/BS (2), Gainesville (1), Gent (1) and Vanves type (1). The frequency of bisalbuminemia was 0.35 per 1,000 in a population of 19,949 blood donors.

IN the method of electrophoresis on starch gel recommended by Smithies1, the gels obtained after the electrophoresis and staining of proteins are opaque, difficult to preserve and not suitable for photometric evaluation. For this reason we have devised a technique for preparing starch gels which remain transparent after staining.

WITH one-dimensional starch gel electrophoresis, the following experimental procedure allows the separation of human plasma into a large number of components.

The method of zone electrophoresis in starch gel enabled Smithies1 to describe three haptoglobin groups in human sera. Using a standard technique previously reported2,3 we have examined the sera of 406 blood donors living in Paris. In each ‘Plexiglas’ tray (internal dimensions 234 mm. × 80 mm. × 6 mm.) three serum samples mixed with a haemoglobin solution (0.05 ml. of a solution containing 50 mgm. of haemoglobin being added to 1 ml. of serum) were allowed to migrate simultaneously, side by side, for 18 hr., under a potential of 100 V. After electrophoresis the starch gels were divided into two slices and stained, one with amido black, and the other with benzidine reagent for peroxidase activity. The technique was the same as that previously described and used to detect haemoglobins in agar gels4 but without using zinc acetate as a solution.

THE azoproteins of Landsteiner were used at the beginning of immunochemical work using modified proteins to study antigen-antibody reactions. These proteins are usually controlled by the well-known quantitative precipitin reaction of Heidelberger.

QUANTITATIVE analysis after starch-gel electrophoresis is usually difficult due to : (1) the diffusion of protein fractions, (2) the difference of migration according to the thickness of the gel, and (3) the fragility and opalescence of this migrating medium.

A micro method for starch gel electrophoresis is described, which has the same resolving power as the original method, but has moreover the following advantages: 1. It is more rapid, since preparing and hardening of the gel takes 20 min; for serum the electrophoresis takes 15 min, and staining plus washing takes 5 min only. 2. It requires a very small sample only (0.2 μl for serum). 3. The gels are small and easy to handle and to store. After the completion of this manuscript, our attention was drawn to the micro electrophoresis method described by Mouray. (H. Mouray, J. Moretti and J.-M. Fine, Bull. Soc. Chim. Biol. , 43 (1961) 993). This method is intermediary between Smithies' macro method and the micro method described above. Mouray, using object glasses, require a migration time of 1 1 2 –2 hours and a sample volume of 5–10 μl.