Chromatographic separation of chicken liver lactate dehydrogenase and xanthine dehydrogenase

Abstract

The purified preparations obtained on treating chicken liver homogenates by a process of thermic denaturation at 75° C and subsequent precipitation with ammonium sulphate (40-60 % saturation) contains jointly LDH and XDH at a degree of purification relative to the original homogenate of 15.5 and 24 times respectively.

The separation of both enzymes from the above purified preparations is achieved by a chromatographic adsorption with DEAE-cellulose. We obtain a XDH concentrated 65.5 times and an extract of LDH, the specific activity of which is 51 times greater than that of the supernatant proceeding from the original homogenate.

The chromatrographic preparations of XDH and LDH show electrophoretically a number of bands much inferior to that of the original homogenates (7 proteic bands); in the XDH the number of bands is two while for the preparation of LDH there can be detected three proteic bands, the most intense corresponding to the LDH, according to observation on developing the pherograms with the specificic dehydrogenase developer.

The real Km of the chicken liver XDH as determined in our preparation are: Km(xanthine) = 1.30 X 10-5 M (saturanting concentration NAD) and Km(NAD) = 2.85 X 10-5 M (saturanting concentration xanthine). The real constants for the pyruvate- LDH-NADH system are: Km(pyruvate) = 4.87 X 10-5 M (saturanting concentration NADH) and Km(NADH) = 1.09 X 10-5 M (saturanting concentration pyruvate). The stated values are in accordance with those determined for XDH and LDH on utilizing homogenates of chicken liver gland.