Heat-induced quaternary transitions in hetero- and homo-polymers of alpha-crystallin

Purpose: To compare the effects of heat incubation on the structure and fun ction of native alpha -crystallin, urea denatured/ renatured alpha -crystal lin, and alphaA and alphaB-crystallin homo-polymers purified from bovine le nses. Methods: Each of the alpha -crystallin samples were incubated for 1 h at te mperatures ranging from 35 degreesC to 70 degreesC. After heat incubation s tructural perturbations in each of the samples were studied using non-denat uring gel electrophoresis, transmission electron microscopy (TEM) and far-U V circular dichroism. The chaperone-like activity of each of the heat-treat ed samples was measured using the DTT induced insulin aggregation assay. Results: The native alpha -crystallin samples showed secondary structure pe rturbations, an increase in aggregate size and asymmetry, and an increase i n chaperone-like activity after heat incubation above 50 degreesC. The othe r three sample types showed secondary structure perturbations beginning at lower incubation temperatures, and a progressive decrease in chaperone-like activity with exposure to increasing temperatures. TEM showed all samples formed large asymmetric high molecular weight aggregates after incubation a t 65 degreesC. Conclusions: The urea denaturation/renaturation of alpha -crystallin has be en shown to result in the loss of a small amount of alpha -helix, but to ha ve no effect on chaperone-like activity under standard test conditions. The present results indicate this lost alpha -helix may be responsible for the differential effects of heat incubation on the different forms of alpha -c rystallin.