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.

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A device for preparative separation of substances according to their molecular size and electric charge in free solution was designed.
It consists of several chambers separated by semipermeable membrane (Millipore filter). A sample solution is applied in one of the chambers and electrophoresis is performed in vertical position with cooling.
Successful separation of serum albumin and γ-globulin was made by the apparatus.
Characteristics of the device are as follows:
1) Separation ability can be increased with molecular sieve effect by choosing a suitable pore size of membranes.
2) The procedures are quite simple since the sample is applied and the fractions after separation are collected in the form of free solution.
3) The electroendosmotic flow can be eliminated by hydrostatic pressure induced by vertical positioning of the chambers.
4) The applicable amount of the sample is in the range of 0.5 to 1.2g of protein.

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