Jornal de Oftalmologia Clínica e Experimental
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Amplified Chromatographic Signal and UV-Absorbance Spectroscopy to Evaluate Lysozyme after Thermal Denaturation and in Contact Lens Extracts

Claude J Giasson, Danielle Béland and Langis Michaud

Objective: Amplify the absorbance signal related to the lysozyme extracted from contact lenses as measured by high-performance liquid chromatography (HPLC); examine the purity of lysozyme and how much thermal denaturation affects its elution profile.
Methods: In trial chromatographic runs during which contact lens extracts had been injected into the system, the fractions collected between 4 and 5.5 minutes pointed to lysozyme as the eluting protein as indicated by a Western blot. Proteins were extracted from contact lenses in a 50:50 solution of 0.2% trifluoroacetic acid (TFA): acetonitrile (ACN). Each contact lens extract was separated into 2 aliquots. After vacuum evaporation, aliquots no 1 were dissolved into the initial mobile phase to produce an enrichment factor of 8. Aliquots no 2 were left untreated in the regular extraction solution. Calibration of the absorbance signal at 220 nm allowed measuring the lysozyme levels in chromatograms. Parallel injections of both aliquots into the HPLC allowed comparing their lysozyme content. Purity of the lysozyme extracted was evaluated by viewing its absorbance spectrum across peaks. Lysozyme solutions, previously heated to 80 and 100°C were injected and compared with control solutions. Differences in median lysozyme content between aliquots no 1 and 2 and in peak area of lysozyme heated compared to control were tested with non-parametric methods.
Results: Median lysozyme level measured in enriched and regular extracts differed significantly (39.8 and 21.5 μg, respectively). However, once corrected for different injection volume and concentrating factor, the mean enriched lysozyme level (21.4 μg) was close to the one found in the regular extract. Observation of spectral absorbance suggests that eluting lysozyme is free from contaminants. Median ratios of peak areas of heated lysozyme over peak area of control lysozyme differed significantly from 1 at the temperature of 100°C (0.91), but not at the one of 80°C (0.95) with the signed rank test. Even when heated at 80°C, the elution profile of lysozyme appeared less symmetrical compared to control and presented an additional inflection point. These subtle changes were increased at 100°C.
Conclusion: Enrichment of the lysozyme extracted from contact lenses and solubilized into the initial mobile phase improves the sensitivity of this chromatographic procedure. This chromatographic protocol coupled with UV absorbance spectroscopy can detect thermal denaturation at 80 and 100°C.