Primary structure of the abundant seed albumin of Theobroma cacao by mass spectrometry.
The most abundant albumin present in seeds of Theobroma cacao was purified to apparent homogeneity as judged by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH(2)-terminal sequence analysis. Tryptic peptide mass fingerprinting of the purified protein by HPLC/ESI-MS showed the presence of 16 masses that matched the expected tryptic peptides corresponding to 95% of the translated amino acid sequence from the cDNA of the 21 kDa cocoa albumin. Collision-induced dissociation MS/MS analysis of the C-terminal peptide isolated from the CNBr cleavage products provided unequivocal evidence that the mature cocoa albumin protein is nine amino acid residues shorter than expected from the reported cDNA of its corresponding gene. The experimentally determined M(r) value of 20234 was in excellent agreement with the truncated version of the amino acid sequence. The purified cocoa albumin inhibited the catalytic activities of bovine trypsin and chymotrypsin. The inhibition was stoichiometric with 1 mol of trypsin or chymotrypsin being inhibited by 1 mol of inhibitor with apparent dissociation constants (K(i)) of 9.5 x 10(-8) and 2. 3 x 10(-6) M, respectively, for inhibitor binding at pH 8.5 and 37 degrees C. No inhibition of the catalytic activities of subtilisin, papain, pepsin, and cocoa endoproteases was detected under their optimal reaction conditions.
Kochhar
S
S
Nestlé Research Center, P.O. Box 44, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland. sunil.kochhar@rdls.nestle.com
Gartenmann
K
K
Juillerat
M A
MA
D000418:Albumins / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002099:Cacao / Q000737:chemistry*; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000378:metabolism
D000418:Albumins; D010446:Peptide Fragments; D010940:Plant Proteins; D011480:Protease Inhibitors; D002918:Chymotrypsin; D014357:Trypsin