Kiyoko Sano

Automatic assay of urinary protein using Coomassie Brilliant Blue G-250

Abstract An automatic method for the protein assay using Coomassie Brilliant Blue G-250 was developed and applied to the assay of urinary proteins. In developing the automatic system, the adhesion of protein-bound dye to the walls of the flow cell and tubes was found to be the most troublesome problem, by which the baseline was shifted upwardly to give positive errors. For the purpose of preventing such adhesion, the concentration of CBB was reduced to half of that used in the manual method, glass tubes and glass coils were changed to those made of Kel-F material, and the flow cell was coated with fluorine resin. As a result, the staining with protein-bound dye was nearly completely eliminated. The final system showed satisfactory ability in performance, namely, the value of a coefficient variation for the reproducibility within run was 1.3%, that for the carry over was 0–1.1%, and the recovery was 98.8%. The calibration curve was linear in a range of 0–1000 μg/ml, and 80 samples could be processed in 1 h. Thus, the present method may serve as an efficient automatic protein analyzer for routine clinical tests of urine samples.

A new method of collecting saliva from human palatine glands for electrophoretic study.

Abstract After stimulating the surface of palatine mucosa by applying 0.2 per cent ZnCl 2 solution, 20–30 μl of saliva secreted from the openings of the palatine gland was directly collected using a newly-developed sampler. Saliva collected with the sampler gave 12 well-defined protein bands on SDS electrophoresis, whereas saliva obtained by a conventional filter-paper disc technique gave fewer and less dense protein bands.

Analysis of human palatine salivary proteins by isoelectric focusing in agarose

An electrophoretic method capable of analysing a small volume (5-10 microliter) of human palatine saliva without prior concentration was developed by combining the high resolution of isoelectric focusing in agarose with a highly sensitive silver protein staining method. Human palatine saliva exhibited 23 protein bands, those at pI 4.3-4.4, 4.7, 5.3 and 5.8 being major bands.

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.