Liberation of tryptic fragments from caseinomacropeptide of bovine x-casein involved in platelet function

Carbohydrate-free caseinomacropeptide (CMP) was purified from rennet-hydrolysed caseinate by trichloroacetic acid precipitation and DEAE-TSK Fractogel-650 ion-exchange chromatography. To study the liberation of 106-112, 106-116 and 113-116 fragments from carbohydrate-free CMP involved in platelet function, a quantitative study was made on the rate of hydrolysis of the three peptidic bonds that are susceptible to the action of trypsin. Data were obtained from reversephase (Ultrabase column) and cationic-exchange (Mono S column) h.p.l.c. On the basis of the disappearance of substrate, keat and K. were respectively 3.95 s-' and 0.2 mm. The two 111-112 and 112-113 bonds were split according to similar kinetic parameters (kcat = 1.97 s-1, Km = 0.2 mM) and much faster than the 116-117 bond. The difference in susceptibility of the bonds can probably be attributed to the nature of residues flanking the primary proteolytic sites rather than to their accessibility to the proteinase. On the basis of our results the 106-116 fragment cannot be formed.