An enzymatic estimation of free hydroxyproline in tissue hydrolysates

An enzymatic method using 4-hydroxyproline 2-epimerase (EC 5.1.1.9), D-amino acid oxidase (EC 1.4.3.3.), and a colorimetric reagent of the mixture of Ti(IV) and 4-(2-pyridylazo)-resorcinol was found to be useful in the spectrophotometric assay for hydroxyproline in collagenous tissue hydrolysates. It was possible to determine free hydroxyproline in 250 microliter of tissue hydrolysates at concentrations ranging from 1 to 12.5 micrograms. The coefficient of variation was less than 3%, and the correlation between the proposed method and the colorimetric method (K.I. Kivirikko, O. Laitinen, and D. J. Prockop (1967) Anal. Biochem. 19, 249-255) was 0.973. Thus, this method is convenient because it requires less time than the above colorimetric method, and is also reproducible and sensitive.