Joji Hori

Precipitation of bovine serum albumin by thiocyanate ion.

The ability of anions to precipitate bovine serum albumin (BSA) at pH 4.0 increased in the order, chloride < bromide < nitrate < thiocyanate = iodide. Using the most effective anion, thiocyanate, details of precipitation were studied. It was found at pH 4.0 that the precipitate that formed below 20 ° and that formed above 20 ° had different properties; the former dissolved in distilled water, but the latter did not. The amount of precipitate in the pH range 3.5–4.5 was different depending upon the pH value at the two temperatures, 0 ° and 25 °; with increase in pH, the percentage of BSA precipitating decreased. The interpretation was made that the precipitation is correlated to the isomerization of BSA, N + nH+ F, which takes place in pH range 3.5–4.5, and that only the F form is precipitated. A reaction scheme of the precipitation was proposed with a few assumptions. Reversibility of precipitation and of dissolution of precipitate was observed. Thus the precipitates formed are not irreversibly denatured BSA.

EFFECT OF METALLIC CATIONS ON HUMAN SERUM: STUDY BY STARCH-GEL ELECTROPHORESIS. I. EFFECT OF PB++, CU ++, AND NH4+.

Abstract When Pb(NO 3 ) 2 or CuSO 4 is added to human serum, the amount of precipitate increases with increase in the concentration of the cation. The supernatant was analyzed by starch-gel electrophoresis. Some protein components of human serum are precipitated at lower concentrations and some resist precipitation even at higher concentrations of a cation. The precipitability of a component depends on the nature of the cation. The main components in the supernatant obtained when serum diluted 50% is made 30 m M in Pb ++ , are transferrin and γ-globulin. Almost all the γ-globulin is precipitated when serum diluted 50% is made 6 m M in Cu ++ ; almost all the proteins are precipitated when the final concentration of Cu ++ is 20 m M . There are two interesting phenomena. One is that the zone of a particular component disappears abruptly at a certain concentration of cation, while a new zone appears, and probably indicates that the component is modified by the cation with a change in mobility. The other is that the intensity of staining of a particular component increases with increase in the cationic concentration. An explanation is that the metallic cation (Pb ++ or Cu ++ ) bound to the protein binds dye (e.g. protein − —Pb ++ —dye − ). Human serum albumin recrystallized three or four times by ammonium sulfate salting-out was analyzed by starch-gel electrophoresis. There were four zones besides the albumin zone. The pattern is similar to that obtained from human serum albumin prepared by cold ethanol fractionation. Human serum albumin extracted from the albumin zone of “starch-block” electrophoresis was shown by starch-gel electrophoresis to be more homogeneous than albumin prepared by salting-out.

Some observations on the isomerization of horse and human serum albumins

Abstract Electrophoretic studies were made of normal horse and human serum albumins over the pH range 3.6–6.8 and at 25 °. The ionic strength of acetate or phosphate buffer used was 0.1, and the concentration of protein was 0.5% (g./100 ml.). Patterns were usually enantiographic and there were two (N and F 1 ) or three (N, F 1 , and F 2 ) boundaries in the pH range 3.6–5.2. The areas of the N and F 1 boundaries changed continuously with pH, and the area of F 2 was almost constant. The results were interpreted, in the same way as was previously done in the case of bovine serum albumin, by the isomerization equilibrium: N + 2H + ai F. Thus the NF isomerization equilibrium exists at least in three albumins: horse, human, and bovine serum albumins. There are two ways of interpretation of the F 2 boundary which appeared at ionic strength 0.1. One is that the F form consists essentially of the two forms F 1 and F 2 . The other is that F 2 is a reaction boundary; this boundary is formed in the electrophoretic process by a reaction between the F form and the supporting medium. The area composition of the F 2 form was only ca . 5%. Thus even if the F 2 boundary is excluded in the calculation of the isomerization curve, the position of the curve is not seriously affected. The isomerization curve of an aged horse serum albumin was determined. There was a plateau in the isomerization curve, as in the case of bovine serum albumin in the presence of 2 M urea. Thus the appearance of a plateau in the isomerization curve would be associated with a prerequisite (first step) of denaturation.

A method for the rapid elution of proteins from "mashed gel" after starch gel electrophoresis.

Abstract An improved method for the elution of serum proteins from starch gel sections through secondary electric fields after electrophoresis is described. By using gel sections that had been mashed to an appropriate extent in the second run, almost complete recovery of the proteins was obtained rapidly, except for γ-globulin. The effect of “mashed” on the recovery of albumin was investigated by varying the degree of mashing.

A double-stage type apparatus for vertical gel electrophoresis

Abstract An apparatus described here has the feature that not only vertical electrophoresis, either on starch or polyacrylamide gel, of a large number of samples can be done simultaneously within a short time, but that thin-layer or micro starch gel electrophoresis can be carried out simply. Two gel trays are attached to the apparatus, serially connected with interposed buffer vessels, and electrical contact between gel and buffer is made by a specially designed bridge. The advantages resulting from the use of this bridge are described.

Improved methods of molding starch gel for zone electrophoresis

Abstract Two improved methods are described for molding starch gel with no distortion in vertical zonal profiles of electrophoresed substrates. Each method is applicable to preparation of the original thick-layer gel as well as of the thin-layer gel by a simple technique, and is useful for qualitative and quantitative estimations of apparent distribution of components that penetrate the gel. The results of preliminary considerations of the main causes of distortion of zones are also described.

Alkaline denaturation of bovine serum albumin III. Effect of palmitic acid.

The complexes APν (A: bovine serum albumin (BSA), P: palmitic acid, ν: 0.63-8.1) were exposed to high pHs for various periods (15min.-7h) at 20°C. The results obtained by the quantitative analysis of disc gel electrophoresis are as follows: 1. When the complexes were exposed to pH 11.3 for 1h, the BSA containing 6-8mols of palmitic acid per mol of albumin (AP6-AP8) did not cause the “first reaction” and was not denatured at all. 2. With further increase in exposure pH and/or exposure time, the “first reaction” progressed over various compositions of the complex. Namely, there was no resistant form to alkaline denaturation under these conditions of exposure; nevertheless, AP6-AP8 revealed the higher stability than any other complex against the denaturation.