Abstract
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 degreesC to 70 degreesC. 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 degreesC. 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 degreesC. 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.
| Original language | English |
|---|---|
| Pages (from-to) | 228 - 233 |
| Number of pages | 6 |
| Journal | Molecular Vision |
| Volume | 7 |
| Issue number | 32 |
| Publication status | Published - 2001 |