FRACTIONS FROM COMMERCIAL COLLAGENASE PREPARATIONS - USE IN ENZYMATICISOLATION OF THE ISLETS OF LANGERHANS FROM PORCINE PANCREAS

Transplantation of isolated islets of Langerhans is an intriguing poss ibility for the treatment of diabetes mellitus. The isolation of islet s from pancreata requires the specific dissociation of the tissue. Com mercial collagenases from Clostridium histolyticum are widely used for this purpose. Unfortunately, the effectiveness of these commercial en zymes is not predictable and differs considerably between suppliers an d even from lot to lot. This is due mainly to differences in their spe cific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants. Free flow zone electrophoresis (FFZ E) was used to separate the effective protein components from undesire d compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities. Fractionation of crude collagenases b y FFZE resulted in partially purified protein fractions that were enri ched for collagenase and tryptic activities, and contained only trace amounts of neutral protease. These preparations proved to be highly ef fective in an in vitro assay for the liberation of viable islets from porcine pancreas. To scale up the production of these collagenases wit h defined enzyme composition, we fractionated two different lots of a commercial collagenase from C. histolyticum (one lot effective in isle t isolation, the other not) by using fast protein liquid chromatograph y (FPLC) on hydroxyapatite. Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collage nase activities and low levels of neutral protease. The chromatographi c protocol developed in this study converted a non-effective collagena se lot into a preparation that allowed successful islet isolation.