Purification of the homogentisic acid oxidase from mammalian liver

Abstract

Alkaptonuria is a rare human autosomal recessive disease caused by the lack of homogentisic acid oxidase (HTO) activity. This enzyme, produced mainly in liver, has been purified and characterized from bovine liver. The aim of this work was to attempt a new purification approach for this enzyme from rabbit liver and to compare its characteristics with the enzyme isolated from human embryonal liver and livers of mice suffering with alkaptonuria. The crude enzyme extracts from livers of rabbit, human embryo and alkaptonuric mice were purified on Sephadex G-100, DEAE-cellulose and sucrose gradients. The pH and temperature optima were determined, as was electrophoretic mobility on SDS-P. We show that HTO activity was completely absent in the liver of the alkaptonuric mouse. In contrast, we purified HTO from rabbit liver 54-fold and determined some basic biochemical characteristics of this enzyme: the pH optimum was approximately 5 and the temperature optimum was 30°C. Although the molecular mass of the enzyme was estimated to be 450–480 kDa, it could be degraded to two proteins with Mw 230 and 200 kDa. The K M of rabbit HTO was estimated to be 1 × 10 −5 M and the activity of HTO was decreased by the salt. Similar biochemical features were obtained with HTO obtained from the human embryo and normal mouse liver. In this study, we biochemically characterized HTO from rabbit liver. We show the inhibitory effect of salt on HTO activity. While this protein could be found in normal murine liver and human embryonal liver, the liver of alkaptonuric mice completely lacked HTO activity.