o,o-Dityrosine in native and horseradish peroxidase-activated galactose oxidase

Treatment of galactose oxidase with catalytic amounts of horseradish peroxidase results in increases in both enzyme activity and Cu(II)-associated absorbance. This reaction requires O₂ and is reversed upon removal of O₂ or peroxidase. o,o-Dityrosine is detected in amino acid hydrolysates of peroxidase-treated galactose oxidase as a ninhydrin peak. Furthermore, even native enzyme contains this species as detected by fluorescence measurements. Peroxidase treatment increases the amount of dityrosine present. The dityrosine forms an intramolecular crosslink, the first such crosslink found in a nonstructural protein. The peroxidase-catalyzed formation of the dityrosine and putative precursor radical(s) is thought to involve a tyrosyl ligand to the Cu(II) in galactose oxidase. Such a radical may be involved in the activation observed.