September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Properties of Alpha Crystallin under High Hydrostatic Pressure
Author Affiliations & Notes
  • Joseph Horwitz
    Jules Stein Eye Institute, UCLA, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Joseph Horwitz, None
  • Footnotes
    Support  NIH Grant EY023588
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 727. doi:
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      Joseph Horwitz; Properties of Alpha Crystallin under High Hydrostatic Pressure. Invest. Ophthalmol. Vis. Sci. 2016;57(12):727.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : To study the structural properties of alpha crystallin under high hydrostatic pressure. Pressure perturbation is completely distinct from thermal, or chemical denaturation that uses molar concentrations of urea or guanidine hydrochloride. A major advantage of pressure perturbation is that in most cases the unfolding of proteins is a reversible process, which allows for useful thermodynamic determinations. Two previous papers of high hydrostatic pressure of alpha crystallin gave conflicting results. Bode et al (Biochem J, 2003: p370) claims that alpha crystallin subunits dissociated significantly at pressure of 1000 bars and higher. Skouri-Panet et al (BBA 1764, 2006: p372) showed that with increased pressure the molecular weight of alpha crystallin increased significantly. We revisited the properties of alpha crystallins using high pressure circular dichroism (CD) and scattering techniques.

Methods : To date high hydrostatic pressure CD measurement were impossible to perform. We have developed a novel CD system for measuring the secondary and tertiary structure of protein at high hydrostatic pressure. The same system was also modified to perform 90° scattering measurement at various hydrostatic pressure.

Results : We found that the discrepancy between two papers cited above is because of pressure effect on the pH of the buffer used. Skouri-Panet et al used phosphate buffer. At 2000 bars the pH will drop from 7 to 6 and under this condition alpha crystallin will become much bigger. Bode et al used BES buffer, which is significantly less sensitive to pressure effects. However, under this condition we find that the quaternary structure of alpha crystallin is stable with relatively little dissociation. At pressure up to 2000 bars CD measurements show that the secondary structure of alpha crystallin is very stable as a function of pressure. The properties of recombinant alpha A and alpha B were compared to native bovine alpha crystallin. Data of some mutations of alpha crystallin will be presented.

Conclusions : High hydrostatic pressure spectroscopy can provide useful new information about the physico-chemical properties of alpha crystallin.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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