Molecular characterization of peptides released from beta-lactoglobulin and alpha-lactalbumin via cardosins A and B.
Crude mixtures of aspartic proteases from flowers of the plant Cynara cardunculus have been studied frequently, as have activities of such enzymes (in pure form) on caseins from bovine, ovine, and caprine sources. This research study addressed pure bovine whey protein as substrates; that is, alpha-lactalbumin (alphaLA) and beta-lactoglobulin (alpha-LG), submitted to hydrolysis by 1 of 2 aspartic proteases (cardosins A and B), previously extracted and purified from C. cardunculus. Samples collected, following incubation at 55 degrees C and pH 5.2, were assayed by fast protein liquid chromatography, reversed phase-high performance liquid chromatography, and tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis; the major peptides released were then collected and sequenced by Edman degradation. Cardosin B and, to a lesser degree, cardosin A showed proteolytic activity toward alpha-LA, but the hydrolyzates produced were characterized by distinct peptide profiles. Cardosin B possesses a broad specificity, and produces several hydrophobic peptides (at least 5, with molecular mass in the range 2 to 8 kDa) in the early stages, which eventually become more hydrophilic (with molecular mass below 2 kDa) at later stages of hydrolysis. Cardosin A was found to cleave alpha-LA at the peptide bonds Phe28-Arg29, Gly54-Tyr55, Ala59-Ile60, Leu71-Phe72, and Leu105-Thr106, whereas cardosin B cleaved Ala19-Glu20, Phe28-Arg29, Glu30-Leu31, Tyr37-Gly38, Trp45-Val46, Phe50-His51, Ala59-Ile60, Ser66-Thr67, Leu71-Phe72, Phe72-Gln73, Gln73-Ile74, Ile78-Trp79, Leu115-Asp116, and Leu124-Ala125. Conversely, cardosins A and B are apparently not active on beta-LG.
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Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, P-4200-072 Porto, Portugal.
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D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007768:Lactalbumin / Q000378:metabolism*; D007782:Lactoglobulins / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D007782:Lactoglobulins; D010446:Peptide Fragments; D010940:Plant Proteins; D007768:Lactalbumin; D016282:Aspartic Acid Endopeptidases; C097455:aspartic proteinases, Cynara cardunculus