Bovine α-lactalbumin (αLA) has four disulfide (SS) bonds in the native form (N). On the oxidative folding pathways of this protein, two specific SS folding intermediates, i.e., (61-77, 73-91) and des[6-120], which have two and three native SS bonds, respectively, accumulate predominantly in the presence of Ca2+. In this study, we reinvestigated the pathways using a water-soluble cyclic selenoxide reagent, trans-3,4-dihydroxyselenolane oxide (DHSox), as a strong and quantitative oxidant to oxidize the fully reduced form (R). In the presence of ethylenediaminetetraacetic acid (EDTA) (under a metal-free condition), SS formation randomly proceeded, and N did not regenerate. On the other hand, two specific SS intermediates transiently generated in the presence of Ca2+. These intermediates could be assigned to (61-77, 73-91) and des[6-120] having two common SS bonds, i.e., Cys61-Cys77 and Cys73-Cys91, near the calcium binding pocket of the β-sheet domain. Much faster folding to N was observed in the presence of Mn2+, whereas Na⁺, K⁺, Mg2+, and Zn2+ did not affect the pathways. The two key intermediates were susceptible to temperature and a denaturant. The oxidative folding pathways revealed were significantly different from those of hen egg white lysozyme, which has the same SS-bonding pattern as αLA, suggesting that the folding pathways of SS-containing proteins can alter depending on the amino acid sequence and other factors, even when the SS-bond topologies are similar to each other.
Keywords: calcium binding site; disulfide bond; folding intermediate; lactalbumin; metal ion; oxidation; protein folding; reduction; selenoxide; temperature effect.