Micropreparation of tissue collagenase fragments of type I collagen in the form of surfactant-peptide complexes and their identification by capillary electrophoresis and partial sequencing.
Combination of standard approaches like pepsin digestion and slab gel electrophoresis with capillary separations allows a relatively easy identification of in vivo occurring collagen fragments. Capillary electrophoresis 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 dodecyl sulfate (SDS). While in the first case peptides move to the cathode in a molecular mass dependent manner, in the second case they move towards anode (also in a molecular mass dependent manner). The profiles obtained by the two approaches resemble mirror images with low molecular mass peptides moving first in the acid background electrolyte while they move last in the presence of SDS. It is proposed that in the capillary electrophoretic separation at pH 2.5 the separation mechanism involves the interaction of the individual peptides with the capillary wall while in the second case (pH 4.5) the leading mechanism of separation involves the interaction of the analytes with the micellar phase. For micellar phase separation the system must be run at reversed polarity. Capillary electrophoretic separation in the pH 2.5 buffer is considerably affected by the presence of SDS in the previous steps of peptide preparation. If the peptides are obtained from SDS slab gel electrophoresis, their movement in the capillary electrophoresis step is about three times faster that the movement of corresponding peptides which have not been complexed with SDS.
Deyl
Z
Z
Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Novotná
J
J
Miksík
I
I
Herget
J
J
D000818:Animals; D003094:Collagen / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism*; D003488:Cyanogen Bromide / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D000860:Hypoxia / Q000378:metabolism; D015151:Immunoblotting; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D011651:Pulmonary Artery / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar
D010446:Peptide Fragments; D003094:Collagen; D017364:Collagenases; D003488:Cyanogen Bromide