alpha A-Crystallin high-molecular-weight (HMW) aggregates were prepared by preheating at 80-90 degrees C and studied using spectroscopic measurements. Conformational differences were suggested based on data of increased bis-ANS (4,4(')-dianilino-1,1(')-binaphthalene-5,5(')-disulfonic acid) and ThT (thioflavin T) fluorescence as well as increased far-UV and decreased near-UV circular dichroism (CD). These results indicated that HMW aggregated alpha-crystallin was more hydrophobic than the native alpha-crystallin, possibly resulting from partial unfolding of alpha-crystallin. The two cysteines in alpha A-crystallin were mostly oxidized in HMW aggregates. The effects of HMW aggregation on the dynamic structure were studied with fluorescence resonance energy transfer; subunit exchange became slower. These results strongly suggest that HMW alpha A-crystallin aggregates result from exposure of buried beta-pleated sheets and increased hydrophobic interaction.