Complete protection by α-crystallin of lens sorbitol dehydrogenase undergoing thermal stress

Abstract

Sorbitol dehydrogenase (L-iditol:NAD+ 2-oxidoreductase, E.C. 1.1.1.14) (SDH) was significantly protected from thermally induced inactivation and aggregation by bovine lens α-crystallin. An α-crystallin/SDH ratio as low as 1:2 in weight was sufficient to preserve the transparency of the enzyme solution kept for at least 2 h at 55 °C. Moreover, an α-crystallin/SDH ratio of 5:1 (w/w) was sufficient to preserve the enzyme activity fully at 55 °C for at least 40 min. The protection by α-crystallin of SDH activity was essentially unaffected by high ionic strength (i.e. 0.5 M NaCl). On the other hand, the transparency of the protein solution was lost at a high salt concentration because of the precipitation of the α-crystallin/SDH adduct. Magnesium and calcium ions present at millimolar concentrations antagonized the protective action exerted by α-crystallin against the thermally induced inactivation and aggregation of SDH. The lack of protection of α-crystallin against the inactivation of SDH induced at 55 °C by thiol blocking agents or EDTA together with the additive effect of NADH in stabilizing the enzyme in the presence of α-crystallin suggest that functional groups involved in catalysis are freely accessible in SDH while interacting with α-crystallin. Two different adducts between α-crystallin and SDH were isolated by gel filtration chromatography. One adduct was characterized by a high M(r) of approximately 800,000 and carried exclusively inactive SDH. A second adduct, carrying active SDH, had a size consistent with an interaction of the enzyme with monomers or low M(r) aggregates of α-crystallin. Even though it had a reduced efficiency with respect to α-crystallin, bovine serum albumin was shown to mimic the chaperone-like activity of α-crystallin in protecting SDH from thermal denaturation. These findings suggest that the multimeric structural organization of α-crystallin may not be a necessary requirement for the stabilization of the enzyme activity.