Hydroxyproline Assay Kit (Perchlorate-Free)

Hydroxyproline ((2S,4R)-4-hydroxyproline, Hyp) is a nonproteinogenic amino acid formed by the enzyme prolyl-4-hydroxylase, which catalizes the post-translational modification of polypeptide proline residues following protein synthesis. In animals, hydroxyproline is found almost entirely in collagen, which contains approximately 12-14% hydroxyproline by mass (the fibrilous protein elastin also contains small quantities of hydroxyproline). Hydroxyproline in tissue hydrolysates is thus a direct measure of the amount of collagen present. Collagen is the most abundant protein in mammals and many diseases are believed to affect collagen turnover, including tumor invasion and metastasis, rheumatoid arthritis, cardiopulmonary fibrosis and muscular dystrophy. The classical assay for determination of Hyp is based upon oxidation of Hyp to a pyrrole intermediate, followed by reaction with Ehrlich’s reagent dissolved in concentrated perchloric acid (HClO4). Perchloric acid is a hazardous material that is both toxic and highly reactive, requiring special handling and waste-disposal protocols. BioVision’s Hydroxyproline Assay Kit II (Perchlorate-Free) uses a proprietary acidic developer solution to accurately measure Hyp in tissue and protein/peptidase hydrolysates without the use of hazardous perchlorates. It is a quick and convenient protocol, yielding a brightly-colored chromophore with an absorbance peak at 560 nm. The assay can detect a minimum of 0.05 µg hydroxyproline per well in a 96-well format and generates colorimetric results that are virtually identical to the classical perchloric acid-based method.