Multiple forms of human 4-hydroxyphenylpyruvate dioxygenase (II).

Purified human liver 4-hydroxyphenylpyruvate dioxygenase (4-hydroxyphenylpyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating), EC 1.13.11.27) has been resolved into three major forms (1, 2. and 3) by triethylaminoethyl cellulose chromatography, agarose electrophoresis, and isoelectric focusing. Electrophoretically homogenous preparations of the major enzyme forms were obtained by rechromatography on triethylaminoethyl cellulose and electrofocusing in pH range 6 to 8. Two minor enzyme forms (minor low pI and minor high ~1) were detected by isoelectric focusing. The protein in the minor forms was less than 5% of that in the major forms. The three major enzyme forms showed immunological identity and had the same ratio between the activity with 4. hydroxyphenylpyruvate and phenylpyruvate as substrate. Their isoelectric points differed by about 0.3 pH units between pH 6.5 to 7.5. The enzyme forms had the same mass as determined by gel filtration in nondenaturing media and the same subunit mass as determined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, 43 kilodaltons. The multiple forms were not interconvertible by oxidation, reduction, pyridine inactivation, metal chelator treatment, or treatment with neuraminidase. No NH,-terminal residue was found. The major enzyme forms had about the same Michaelis constants, 0.03 mu. The apparent maximal velocities and the susceptibilities to inhibition by 4hydroxyphenylpyruvate were different. Enzyme Form 2 was resolved into two protein bands, spaced about 0.1 pH unit apart, at polyaerylamide-stabilized isoelectric focusing in urea. Enzyme Form 1 contained only one of these bands and enzyme Form 3 the other. The major forms of human liver 4hydroxyphenylpyruvate dioxygenase thus appear to be dimerit combinations of two subunits differing in charge but not in size.