Hiroko Cho

Re-evaluation of normal human urinary proteins fractionated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Proteins in normal human urine were clearly fractionated into 26 bands with molecular weights from 14,000 to 230,000 by means of one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with silver staining. The main band contained uromucoid, and the second main band had albumin. However, when urine samples from healthy persons were electrophoresed in the absence of SDS using polyacrylamide gel or agarose gel, or a cellulose acetate membrane, albumin but not uromucoid, frequently formed the main protein band. It is suggested that this is due to the complexing of uromucoid subunits to form a large molecule which cannot penetrate into the gel. In order to correctly fractionate all the proteins contained in normal human urine, it was concluded that it was best to treat a urine sample with SDS with pre-condensation, fractionate it by SDS-PAGE and stain fractionated proteins by a highly sensitive method such as silver staining.

Study on heterogeneity of serum γ-glutamyltranspeptidase in normal subjects and patients with hepatobiliary diseases

Heterogeneity of serum γ-glutamyltranspeptidase (GTP) of 23 normal subjects and 82 patients with hepatobiliary diseases were studied by electrophoresis on “Cellogel”.Nine fractions in total were detected in normal and diseased groups. Enzymograms of normal controls were classified into the following three types; 1) a single broad band between albumin and α2 globulin (52.2%), 2) a single sharp band at α1-α2 globulin (39.1%), 3) double bands at albumin-α1 globulin and α1-α2 globulin (8.7%).Enzymograms of patients with hepatobiliary diseases were different from those of normal control.The fractions were classified to main and sub fraction according to density of bands and frequency of appearance of individual fraction as main or sub fraction in hepatobiliary diseases was investigated.Characteristic features of enzymograms in various hepatobiliary diseases were demonstrated and clinical significance of several fractions which appeared frequently were discussed.

Electrophoretic separation of γ-glutamyl transpeptidase isoenzymes on cellulose acetate “Cellogel”

A method was described for separation of γ-glutamyl transpeptidase (γ-GTP) isoenzymes by cellulose acetate membrane (Cellogel). Enzyme activity staining was carried out by use of γ-L-glutamyl-α-naphthylamide as substrate and fast blue B as color developing agent. Zymogram on Cellogel membrane was treated with 5% trichloroacetic acid solution and applied for densitometry.The conditions for this method (concentration of substrate, diazo reagent, glycylglycine, pH of buffer, incubation time, etc.) were examined and optimal conditions were proposed.This method was simple and sensitive enough to detect γ-GTP isoenzyme patterns from the normal serum containing 30mU/ml activity. Linear relationship between color development of zymogram and enzyme activity was also obtained from 30mU/ml to 230mU/ml in serum sample and this method, therefore, was available for the routine work as semi-quantitative method.