Photoexcitation of tryptophan groups induces reduction of two disulfide bonds in goat alpha-lactalbumin.
Illumination of goat alpha-lactalbumin (GLA) with 280 or 295 nm light results in tryptophan-mediated photolysis of disulfide bonds within the protein. The photolysis is not dependent on the absence or presence of Ca(2+) and is observed as well on illumination of native and of partially unfolded GLA. However, photolysis of native GLA results in a partial unfolding of the protein. The latter phenomenon is most clearly observed on fluorescence measurements at low temperatures (near 3 degrees C). The photolysis induces some dimerization and oligomerization, but most GLA molecules remain monomeric. To obtain more information about the reaction products, the illuminated protein is treated with iodoacetamide to label the free thiol groups, it is fragmented with trypsin, and the fragments are analyzed by mass spectrometry. Via this approach, we observe that the cleavage of disulfide bonds is restricted to Cys6-Cys120 and Cys73-Cys91 bonds. The photolytic cleavage of either of these disulfide bonds results in the formation of a single free thiol, a phenomenon restricted to Cys120 and Cys91, respectively. We also found indications that a thioether linkage is formed between Cys73 and Trp60. The alkylsulfenylation of Trp60 presumably results from a combination of primary thiyl and tryptyl radicals.
Interdisciplinary Research Center, Katholieke Universiteit Leuven Campus Kortrijk, B-8500 Kortrijk, Belgium.
Ann
A
Vanhooren
Bart
B
Devreese
Kristien
K
Vanhee
Jozef
J
Van Beeumen
Ignace
I
Hanssens
D000818:Animals; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry* / Q000528:radiation effects; D006041:Goats; D006868:Hydrolysis / Q000528:radiation effects; D007768:Lactalbumin / Q000737:chemistry* / Q000528:radiation effects; D010084:Oxidation-Reduction / Q000528:radiation effects; D010782:Photolysis; D017785:Photons; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014364:Tryptophan / Q000737:chemistry* / Q000528:radiation effects
D004220:Disulfides; D014364:Tryptophan; D007768:Lactalbumin