Vanhooren A, De Vriendt K, Devreese B, Chedad A, Sterling A, Van Dael H, Van Beeumen J, Hanssens I
Interdisciplinary Research Center, Katholieke Universiteit Leuven Campus Kortrijk, Kortrijk, Belgium.
Biochemistry. 2006 Feb 21;45(7):2085-93. doi: 10.1021/bi0517638.
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.
山羊α-乳白蛋白（GLA）含有四个色氨酸（Trp）残基和四个二硫键。用近紫外光照射会导致二硫键断裂并形成游离巯基。为了获取有关反应产物的信息，将照射后的蛋白质进行氨甲酰甲基化处理，并用胰蛋白酶消化，然后通过质谱分析肽段。检测到了含有半胱氨酸120氨甲酰甲基化（Cys120Cam）、半胱氨酸61氨甲酰甲基化（Cys61Cam）或半胱氨酸91氨甲酰甲基化（Cys91Cam）的肽段，以及两个含有新的半胱氨酸-赖氨酸交联的肽段。其中一个是半胱氨酸6与赖氨酸122交联，而另一个交联要么是半胱氨酸91-赖氨酸79交联，要么是半胱氨酸73-赖氨酸93交联；然而，确切的连接方式无法确定。结果表明，半胱氨酸6-半胱氨酸120、半胱氨酸61-半胱氨酸77和半胱氨酸73-半胱氨酸91二硫键发生了光解。虽然已报道GLA中半胱氨酸6-半胱氨酸120和半胱氨酸73-半胱氨酸91二硫键的光解，但此前未检测到半胱氨酸61-半胱氨酸77二硫键的断裂。为了研究各个色氨酸残基的作用，我们构建了GLA突变体W26F、W60F、W104F和W118F，用苯丙氨酸（Phe）取代单个色氨酸残基。与野生型GLA相比，每个色氨酸残基的取代导致产生的巯基减少，这表明GLA中的每个色氨酸残基都有助于二硫键的光解。特异性由反应产物的性质体现。照射W26F突变体时未检测到半胱氨酸6-半胱氨酸120二硫键的断裂，照射W26F或W104F后也未观察到半胱氨酸73-半胱氨酸91二硫键的断裂。相反，在照射任何一个突变体后，都发现了由半胱氨酸61-半胱氨酸77二硫键断裂产生的半胱氨酸61氨甲酰甲基化。
