Ultraviolet illumination-induced reduction of alpha-lactalbumin disulfide bridges.
Prolonged exposure of Ca(2+)-loaded or Ca(2+)-depleted human alpha-lactalbumin to ultraviolet light (270-290 nm, 1 mW/cm(2), for 2 to 4 h) results in a 10-nm red shift of its tryptophan fluorescence spectrum. Gel chromatography of the UV-illuminated samples reveals two non-native protein forms: (1) a component with a red-shifted tryptophan fluorescence spectrum; and (2) a component with kynurenine-like fluorescent properties. The first component has from 0.6 to 0.9 free DTNB-reactive SH groups per protein molecule, which are absent in the native protein and is characterized by slightly lowered Ca(2+)-affinity (2 x 10(8) M(-1) versus 8 x 10(8) M(-1) for the native protein) and absence of observable thermal transition. The second component corresponds to the protein with photochemically modified tryptophan residues. It is assumed that the UV excitation of tryptophan residue(s) in alpha-lactalbumin is followed by a transfer of electrons to the Sbond;S bonds, resulting in their reduction. Mass spectrometry data obtained for trypsin-fragmented UV-illuminated alpha-lactalbumin with acrylodan-modified free thiol groups reveal the reduction of the 61-77 and 73-91 disulfide bridges. The effect observed has to be taken into account in any UV-region spectral studies of alpha-lactalbumin.
Permyakov
Eugene A
EA
Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia.
Permyakov
Serge E
SE
Deikus
Gintaras Y
GY
Morozova-Roche
Ludmila A
LA
Grishchenko
Valery M
VM
Kalinichenko
Lina P
LP
Uversky
Vladimir N
VN
D002118:Calcium / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D010084:Oxidation-Reduction / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D011487:Protein Conformation; D013696:Temperature; D013816:Thermodynamics; D014466:Ultraviolet Rays
D004220:Disulfides; D007768:Lactalbumin; D002118:Calcium