Human
alpha-crystallins were separated from fetal, young, senile nondiabetic and diabetic
lenses. The effects of aging and
diabetes mellitus were studied by fluorescence measurements, including emission maximum, quantum yield and polarization, using both intrinsic probes (
tryptophan and non-
tryptophan) and extrinsic probes [4-(N-iodoacetoxy)N-methylamino-7-nitrobenz-2-oxa-1,3-diazole (
IANBD) and 6-(p-toluidinyl)naphthalene-2-sulfonate (
TNS)]. Results indicate that diabetic effects (glycation and aggregation) give fluorescence change to a far greater extent than that of aging. This was demonstrated by a large decrease in
tryptophan quantum yield and an increase in non-
tryptophan quantum yield, and also by a decrease in polarization of non-
tryptophan. The sulfhydryl (SH)-specific probe
IANBD shows a blue-shift in emission maximum, a decrease in intensity and an increase in polarization. The hydrophobic probe
TNS shows a decrease in both intensity and polarization. These results suggest that
tryptophan oxidation, mixed
disulfide formation and glycation, as well as other undetected post-translational modifications have partially unfolded the
proteins, making the
protein structure less rigid. The possible effect of this unfolding process is that
proteins become more susceptible to aggregation.