Proteolytic digestion of alpha-lactalbumin: physiological implications.
The kinetics of the partial digestion of bovine alpha-lactalbumin (alpha-LA) by trypsin, alpha-chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of alpha-LA to trypsin and chymotrypsin at 37 and 5 degrees C decrease in the following order: Ca(II)-alpha-LA greater than Zn(II), Ca(II)-alpha-LA greater than apo-alpha-LA. The HPLC digestion patterns of Ca(II)-alpha-LA and Zn(II), Ca(II)-alpha-LA at 5 and 37 degrees C were similar, while the corresponding digestion patterns for apo-alpha-LA were quite different, reflecting the existence of the thermally induced denaturation states of apo-alpha-LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded alpha-LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37 degrees C due to Zn(II)-induced shift of the thermal transition of alpha-LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II)-specific site does not cause any drastic changes in the overall structure of alpha-LA. The acidic form of alpha-LA (at pH 2.2 and 37 degrees C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or alpha-chymotrypsin at neutral pH. Complexation of alpha-LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to alpha-LA may have physiological significance (e.g., for nutritional transport).
Hirai
Y
Y
Department of Chemistry, Ohio State University, Columbus 43210.
Permyakov
E A
EA
Berliner
L J
LJ
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D007700:Kinetics; D007768:Lactalbumin / Q000378:metabolism*; D010434:Pepsin A / Q000378:metabolism; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D007768:Lactalbumin; D002918:Chymotrypsin; D014357:Trypsin; D010434:Pepsin A; D015032:Zinc