A simple assay for determining keratin and collagen degradation in vitro

Abstract

A simple assay measuring degradation of human epidermal keratin and bovine tendon collagen is presented. Insoluble protein substrate (30 mg) was incubated with 1 ml buffer and enzyme sample for 1 h at 37 degrees C, following addition of 1 ml distilled water and removal of the remaining substrate by filtration/centrifugation. The protein content was determined in the filtrate/supernatant by the Lowry method. Keratin was prepared as follows: Freeze-drying, homogenization in a mortar or beetling mill, extraction in 0.9% (w/v) NaCl followed by water and 100% ethanol, drying at 37 degrees C. The assay was tested with pig pepsin, bovine trypsin, and crude extract of fish stomach, demonstrating that these preparations are effective in degrading human epidermal keratin.