Probing the molten globule state of alpha-lactalbumin by limited proteolysis.
Limited proteolysis has been used to probe the partially folded state of bovine alpha-lactalbumin (BLA) at acid pH (A-state) or dissolved in aqueous trifluoroethanol (TFE-state). The sites of proteolytic fission have been determined by isolation of the various BLA fragments and comparison of their N-terminal amino acid sequence and amino acid composition after acid hydrolysis, as well as their molecular mass determined by mass spectrometry, with the known sequence of BLA. Incubation of BLA with pepsin at 20-22 degrees C and pH 2.0 in the presence of 0.1 M NaCl results in very rapid cleavage of the 123-residue chain at peptide bond Ala40-Ile41 and subsequently at Leu52-Phe53, leading to a nicked species of BLA constituted by the two fragments 1-40 and 53-123 cross-linked by the four disulfide bridges of the protein. Much slower proteolytic cleavage occurs at Tyr103-Trp104. The highly helical conformational state acquired by BLA when dissolved in aqueous buffer (pH 7.0) containing 50% (v/v) TFE was probed by the TFE-resistant thermolysin. Proteolytic cleavage occurs at the peptide bond Ala40-Ile41 and much more slowly at Phe80-Leu81. Moreover, the peptide bond Gln2-Leu3 at the N-terminus of the chain is partially cleaved by thermolysin. Conversely, native BLA in a pH 7.0 buffer is rather resistant to proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Polverino de Laureto
P
P
CRIBI Biotechnology Centre, University of Padua, Italy.
De Filippis
V
V
Di Bello
M
M
Zambonin
M
M
Fontana
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D006868:Hydrolysis; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010449:Peptide Mapping; D017510:Protein Folding; D013820:Thermolysin / Q000378:metabolism
D015335:Molecular Probes; D007768:Lactalbumin; D010434:Pepsin A; D013820:Thermolysin