Purpose: To compare the effects of heat incubation on the structure and function of native alpha-crystallin, urea denatured/renatured alpha-crystallin, and alphaA and alphaB-crystallin homo-polymers purified from bovine lenses.

Methods: Each of the alpha-crystallin samples were incubated for 1 h at temperatures ranging from 35 degrees C to 70 degrees C. After heat incubation structural perturbations in each of the samples were studied using non-denaturing gel electrophoresis, transmission electron microscopy (TEM) and far-UV circular dichroism. The chaperone-like activity of each of the heat-treated samples was measured using the DTT induced insulin aggregation assay.

Results: The native alpha-crystallin samples showed secondary structure perturbations, an increase in aggregate size and asymmetry, and an increase in chaperone-like activity after heat incubation above 50 degrees C. The other 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 at 65 degrees C.

Conclusions: The urea denaturation/renaturation of alpha-crystallin has been shown to result in the loss of a small amount of alpha-helix, but to have 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-crystallin.