PREFERENTIAL SITES OF TRYPTIC CLEAVAGE ON THE MAJOR BOVINE CASEINS WITHIN THE MICELLE

Caseins, major proteins in bovine milk, are structured in micelles wit hin which the detailed casein organisation remains unclear. We have us ed limited proteolysis to find the most exposed regions of the caseins within the micelle. Under hydrolysis conditions, only 9% of colloidal calcium and 15.6% of the colloidal inorganic phosphate were removed f rom the micelle after 4 h. The released peptides during tryptic hydrol ysis were separated from the residual micelle by ultracentrifugation. They were subsequently analysed and characterised by reversed-phase HP LC coupled online with electrospray ion source mass spectrometry (ESI- MS). After 4 h of hydrolysis, the beta-casein had completely disappear ed while about 40% of alpha(s1)-casein and 37% of kappa-casein remaine d undiges ted. The initial distribution, i.e., 91% caseins and 9% pept ides prior to hydrolysis, was modified after hydrolysis to give 64% pe ptides remaining within the micelles together with 13% caseins and 22% of the peptides produced were released from the micelles. Among the 6 1 peptides identified, 34 arose from beta-casein, 16 from alpha(s1)-ca sein, 11 from alpha(s2)-casein and none from kappa-casein. For the lat ter casein, the failure to detect peptides was due to its lower concen tration in the large micelles compared with that of the beta and alpha (s1)-caseins. The peptides derived from beta-casein were mainly releas ed from the micelle, while peptides from both alpha(s1)- and alpha(s2) -caseins were preferentially retained within the residual micelles, su ggesting that all the caseins are accessible to trypsin but are differ entially involved in the micellar framework. Our results suggest that alpha(s)-caseins plays significant role in the micellar cohesion via s alt binding with colloidal calcium phosphate and hydrophobic interacti on, with itself and with the Ether caseins. (C) Inra/Elsevier, Paris.