MICROPREPARATION OF TISSUE COLLAGENASE FRAGMENTS OF TYPE-I COLLAGEN IN THE FORM OF SURFACTANT-PEPTIDE COMPLEXES AND THEIR IDENTIFICATION BYCAPILLARY ELECTROPHORESIS AND PARTIAL SEQUENCING

Combination of standard approaches like pepsin digestion and slab gel electrophoresis with capillary separations allows a relatively easy id entification of in vivo occurring collagen fragments. Capillary electr ophoresis can be done either in 25 mM phosphate buffer (pH 2.5) or in a 25 mM phosphate buffer (pH 4.5) made 0.1% with respect to sodium dod ecyl sulfate (SDS). While in the first case peptides move to the catho de in a molecular mass dependent manner, in the second case they move towards anode (also in a molecular mass dependent manner). The profile s obtained by the two approaches resemble mirror images with low molec ular mass peptides moving first in the acid background electrolyte whi le they move last in the presence of SDS. It is proposed that in the c apillary electrophoretic separation at pH 2.5 the separation mechanism involves the interaction of the individual peptides with the capillar y wall while in the second case (pH 4.5) the leading mechanism of sepa ration involves the interaction of the analytes with the micellar phas e. For micellar phase separation the system must be run at reversed po larity. Capillary electrophoretic separation in the pH 2.5 buffer is c onsiderably affected by the presence of SDS in the previous steps of p eptide preparation. If the peptides are obtained from SDS slab gel ele ctrophoresis, their movement in the capillary electrophoresis step is about three times faster that the movement of corresponding peptides w hich have not been complexed with SDS. (C) 1998 Published by Elsevier Science B.V.