Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin.
Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.
Vanhooren
A
A
Interdisciplinary Research Center, Katholieke Universiteit Leuven Campus Kortrijk, Kortrijk, Belgium.
De Vriendt
K
K
Devreese
B
B
Chedad
A
A
Sterling
A
A
Van Dael
H
H
Van Beeumen
J
J
Hanssens
I
I
D000595:Amino Acid Sequence; D000818:Animals; D004220:Disulfides / Q000737:chemistry / Q000528:radiation effects*; D006041:Goats; D007768:Lactalbumin / Q000235:genetics / Q000528:radiation effects*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010782:Photolysis / Q000187:drug effects*; D017354:Point Mutation; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014364:Tryptophan / Q000528:radiation effects*; D014466:Ultraviolet Rays
D004220:Disulfides; D010446:Peptide Fragments; D014364:Tryptophan; D007768:Lactalbumin