Paraformaldehyde fixation and some characteristics of lens proteins as measured by Raman microspectroscopy.

In previous studies (Huizinga et al., 1989; Bot et al., 1989) it was demonstrated that the results of water content measurements of human and rabbit lenses estimated by Raman microspectroscopy were comparable to those using differential scanning calorimetry, nuclear magnetic resonance spectroscopy, dynamic light scattering and the dry-weight-frozensection technique. It was emphasized that Raman microspectroscopy is superior in detecting local variations in water content. It was further demonstrated that fixation of the lenses with a cacodylate buffered paraformaldehyde solution had only minor quantitative effects on the water content. Finally, it was shown that slicing of the llxed lenses further improved local positioning of the Raman probe and enabled Raman analysis of adult and aged human lenses. The role of the conversion of sullhydryl (SH) groups into disulfide (SS) bonds in the formation of high molecular weight (HMW) proteins, and consequently in cataractogenesis, seems well established (East et al., 1978; Ozaki et al., 1983; Spector, 1984; Yu et al., 1985). In addition it was found, in aging mice lenses, that in concert with the decrease in SH-groups the ratio of the tryptophan doublet &,,/I,,,) also decreases, whereas the tyrosine doublet (I855/Is32) remains constant (Ozaki et al., 1983). In a hereditary nuclear cataract in mice (cat strain) a decrease in both the tryptophan and tyrosine doublet was found (Itoh et al., 1983). These declines appeared concomitant with the first clinical signs of nuclear opaciilcation. In view of this evidence it seems likely that the SH to SS conversion and the changes in the ratio of the tryptophan and tyrosine doublets play a role in the formation of HMW proteins and cataractogenesis. Because an important goal of our Raman studies is the analysis of changes in water content and protein composition of aging human lenses and as adult human lenses can only be studied by Raman spectroscopy after paraformaldehyde fixation and subsequent slicing, it was essential to analyze the effects of paraformaldehyde fixation on the SH-groups and the tryptophan and tyrosine doublets. For this study fresh rabbit lenses (Chinchilla strain, 6 months old) were screened along the visual axis using the Raman microspectroscopic procedure described earlier (Gijsbers et al., 1986). Subsequently the lenses were immersed in a 0.08 M cacodylate buffered 1% paraformaldehyde solution (3 10 mOsmo1, pH 7.3). After 1 week the lenses were remeasured. The SH-content was indicated as the ratio &,,,/I,,,, (SH/protein) and the tryptophan doublet (ratio: I,,&,,,) and the tyrosine doublet (ratio: &JIs3.J were indicated according to Ozaki et al. (1983). The differences between the lenses under investigation,