Peracetylated Bovine Carbonic Anhydrase (BCA-Ac₁₈) Is Kinetically More Stable than Native BCA to Sodium Dodecyl Sulfate

Abstract

Bovine carbonic anhydrase (BCA) and its derivative with all lysine groups acetylated (BCA-Ac 18 ) have different stabilities toward denaturation by sodium dodecyl sulfate (SDS). This difference is kinetic: BCA-Ac 18 denatures more slowly than BCA by several orders of magnitude over concentrations of SDS ranging from 2.5 to 10 mM. The rates of renaturation of BCA-Ac 18 are greater than those of BCA, when these proteins are allowed to refold from a denatured state ([SDS] ) 10 mM) to a folded state ([SDS] ) 0.1 to 1.5 mM). On renaturation, the yields of the correctly folded protein (either BCA or BCA-Ac 18 ) decrease with increasing concentration of SDS. At intermediate concentrations of SDS (from 0.7 to 2 mM for BCA, and from 1.5 to 2 mM for BCA-Ac 18 ), both unfolding and refolding of the proteins are too slow to be observed; an alternative processs probably aggregationscompetes with refolding of the denatured proteins at those intermediate concentrations.Because it is experimentally impractical to prove equilibrium, it is not possible to establish whether there is a difference in the thermodynamics of unfolding/refolding between BCA and BCA-Ac 18 .

Differentiation of proteins based on characteristic patterns of association and denaturation in solutions of SDS

“…The shape of the peak of ubiquitin remained invariant with decreasing voltage; this observation suggests that the formation of ubiquitin-SDS aggregate at 0.6 mM SDS is rapid relative to the time scale of the CE separation. In the case of BCA at 3 mM SDS, the area of native BCA peak decreased as the residence time of the protein in the capillary increased; we infer that denaturation of BCA at 3 mM SDS occurs on a time scale similar to that of the CE separation (21).…”

“…We cannot, therefore, determine a concentration of SDS at which the mobility of these proteins begins to change. *In ␤-Lgb, Asp-64, and Val-118 in isoform A are replaced by glycine and alanine, respectively, in isoform B (21,22).…”

“…Refolding is clearly slower than unfolding under these conditions. We have studied BCA independently (21,23) and know that it does refold completely with a time constant of Ϸ10 min when the SDS is removed; we cannot predict the long-term behavior of the other proteins.…”

Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation

“…A4551). Acetylated variants of BLA were prepared using protocols for the acetylation of other proteins (Gitlin et al 2006c). Briefly, solutions of BLA (1.5 mg/mL or 27 mM BLA, 100 mM HEPBS buffer, pH 9.0, 10.0 mL total volume) were allowed to react with various volumes of acetic anhydride (800 mM acetic anhydride in dioxane) ranging from 0 to 300 mL; the pH of the reaction mixture was monitored with a pH electrode and kept at 9.0 by the addition of NaOH.…”

Acetylation of Surface Lysine Groups of a Protein Alters the Organization and Composition of Its Crystal Contacts

“…We used acetic anhydride to acetylate the lysine residues of CA (this procedure is quantitative; see Supporting Information); we refer to crystals of the peracetylated variant as [CA-(NHCOCH 3 ) 18 ] -19 . Capillary electrophoresis confirmed that all 18 residues were acetylated (Figure S1) 16 ; X-ray crystallography allowed us to determine its structure, and, thus, permitted a comparison with the structure of wild-type CA, 6 which we, hereafter, refer to as [CA-(NH 3 + ) 18 ] -3. 4 .…”