Measurement of proteolytic activity in biological materials using 125I-labelled casein [proceedings].

The use of radioactively labelled proteins for studying proteolysis is well established (Schneider et al., 1965), as are techniques for radiolabelling proteins, which have been developed mainly for radioimmunassay procedures (Yalow & Berson, 1966). 1311 has been the favoured isotope for such work, although it has a short half-life (8.04 days). "'I has a longer half-life (60 days) and can be efficiently counted in the I4C channel of a liquid-scintillation counter. The technique using 1251-labelled casein for studying proteolytic activity in microbiological materials, notably sewage and preparations of micro-organisms derived from it, is a simple one. After incubation of 1251-labelled casein with the materials, radiolabelled degradation products can be separated from the undegraded protein by perchloric acid precipitation. Subsequent use of a microcentrifuge enables each assay to be performed in a single reaction vessel on a total volume as little as 0.1 ml, minimizing the need for containment facilities for isotope or micro-organisms. The assay suffers from few disadvantages compared with the alternatives available for these materials, such as gelatin penetration. Sewage-derived micro-organisms at a concentration of 106/ml released sufficient radioactivity from the 1251-labelled casein in 90min for measurement purposes. In theory, the sensitivity of such methods of enzyme assay is limited solely by the specific radioactivity of the radiolabelled substrate; in practice, due to self-irradiation, 1251-labelled casein undergoes decomposition leading to the release of acid-soluble radioactivity. The half-life of this process depends on the specific radioactivity; using a preparation of 1251-labelled casein prepared by the method of Hunter & Greenwood (1962) with the specific radioactivity initially of lOCi/g, the halflife of the bound radioactivity was similar to the isotope half-life, 63 days.