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pH-dependent secondary conformation of bovine lens alpha-crystallin: ATR infrared spectroscopic study with
1Department of Medical Research and Education, Veterans General Hospital Taipei, Taipei, Taiwan, Republic of China. sylin@vghtpe.gov.tw
Background:
In order to simulate the usage of different formulations of ophthalmic solution, the protein conformational changes of lens alpha-crystallin in buffer solutions of different pH were investigated.
Methods:
The secondary structure of bovine lens alpha-crystallin in different Mcllvaine buffer solutions (pH 2.2, 4.0, 6.0, 7.2 and 8.0) was determined by attenuated total reflection (ATR)/Fourier transform infrared (FT-IR) spectrometry with second-derivative technique. The turbidity of alpha-crystallin in different buffer solutions was observed.
Results And Conclusion:
The results indicate that alpha-crystallin exists mainly in beta-sheet structure at 1627-1637 cm-1. The conformational components of alpha-crystallin may be closely similar in pH 7.2 buffer solution and in pH 8.0 buffer solution. Once alpha-crystallin dissolved in pH 6.0 and 4.0 buffer solution, the appearance of a component at 1618 or 1620 cm-1, associated with the presence of intermolecular beta-sheet structure or beta-turn structure or amino acid side chains, implied the denaturation of alpha-crystallin, which was even more marked in pH 4.0 buffer solution. Due to the effect of dissociation and stability of alpha-crystallin in pH 2.2 buffer solution, the secondary structure of the intact alpha-crystallin was difficult to evaluate. This study demonstrates that different pH can vary secondary conformational structure of alpha-crystallin, particularly if the pH is below 6.0. This suggests that the secondary structure of alpha-crystallin in buffer solution exhibits pH-dependent characteristics.