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R. A. Moore, R. L. McKown, R. W. Raab, G. W. Laurie; Stability Analysis of Human Prosecretory Mitogen Lacritin by Circular Dichroism. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5288. doi: https://doi.org/.
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Lacritin is a natural human tear protein that is prosecretory for rat lacrimal acinar (Sanghi et al, J. Mol. Biol. 310:127-139) and human corneal epithelial cells. When applied topically to rabbit eyes, it appears to promote tearing. Deletion analysis has revealed that an amphipathic alpha helical region in lacritin’s C-terminus is necessary for activity (Wang et al, J. Cell Biol. 174:689-700, 2006; Ma et al, Exp. Eye Res, in press). Here we ask what conditions denature this alpha helical structure. This data is important towards the potential development of lacritin as a topical therapeutic for dry eye.
Recombinant lacritin was purified using affinity and ion exchange chromatography. Purity and protein concentration were respectively determined by SDS-PAGE and the BCA protein assay. Spectral analysis of lacritin (0.5 mg/ml) in increasing salt, temperatures, and solvent concentrations was studied using a Jasco J-810 spectroplarimeter with CD Pro Software CDSSTR, CONTINLL, and SELCON3.
In phosphate buffer at 25°C, mature lacritin is approximately 57% helical. In 150 mM NaCl, lacritin is 53% helical. The helical content of lacritin increased with increasing concentrations of the solvent trifluoroethanol (TFE) to a maximum of 85% at 60% TFE. Temperature had surprisingly little effect on lacritin helical structure in phosphate buffer (51% after heating to 100°C for 10 minutes).
In an aqueous environment, mature lacritin exhibits a secondary structure that is approximately 57% alpha helical. The helical structure appears to be stable in 150 mM NaCl and at temperatures up to 100°C. In a hydrophobic environment, the helical content of recombinant lacritin can increase up to 85%. This may facilitate interactions with the binding domain (Ma et al, J. Cell Biol. 174:1097-1106, 2006) in the N-terminus of cell surface syndecan-1.
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