Hydration Properties of the Molecular Chaperone alpha-Crystallin in the Bovine Lens

M. A. Babizhayev^1,2,3*, G. M. Nikolayev⁴, S. N. Goryachev⁴, J. Bours⁵, and R. Martin⁶

¹Innovation Vision Products, Inc., 3511 Silverside Road, Suite 105, County of New Castle, Delaware, USA 19810

²Innovative Vision Products, Inc., Moscow Division, ul. Ivanovskaya 20-74, Moscow 127434, Russia; fax: (7-095) 977-2387; E-mail: markbabizhayev@yahoo.com

³Helmholtz Moscow Research Institute of Eye Diseases, ul. Sadovaya-Chernogryazskaya 14/19, Moscow 103064, Russia; E-mail: markbabizhayev@mail.ru

⁴Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University, Moscow 119992, Russia

⁵Institute of Experimental Ophthalmology, University of Bonn, Sigmund Freud Str. 25 D 53105, Bonn, Germany

⁶Healthy LLC, 10488 N. 119th, PL. Scottsdale, Arizona, 85259 USA

^* To whom correspondence should be addressed.

Received January 21, 2002; Revision received April 8, 2003

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Topographic studies of crystalline fractions from different morphological layers of the young adult bovine lens were conducted. Crystallin profiles were obtained for each lens layer, using thin-layer isoelectric focusing in polyacrylamide gel (IEF). Water soluble (WS) crystallins from the lens equator revealed a separation into HM (high molecular weight) alpha_L-, beta_H-, beta_L-, beta_S-, and gamma-crystallins. The nature of the water insoluble (WI) protein fraction in the separated lens layers reflected the aggregated state of alpha_L-, beta_L-, beta_S-, and gamma-crystallins in different regions of the lens, concealed in the central cavity of the alpha-crystallin chaperone model. The IEF data demonstrate a possible chaperone-like function for alpha-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. The water binding properties of bovine lens alpha-crystallin, calf skin collagen, and bovine serum albumin (BSA) were investigated with various techniques. The water adsorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. The NMR spin-echo technique was used to study the hydration of protein samples and to determine the spin-spin relaxation times (T₂) from the protons of water adsorbed on the proteins. Isolated bovine lenses were sectioned into 11-12 morphological layers (from anterior cortex through nucleus to posterior cortex). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During water vapor uptake at relative humidity P/P₀ = 0.75, alpha-crystallin did not adsorb water suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At relative humidity P/P₀ = 1.0, the adsorption of water by alpha-crystallin was 17% with a single component decay character of spin echo (T₂ = 3 msec). Addition of water to alpha-crystallin to about 50% of its weight/weight in the protein sample showed T₂ = 8 msec with only one single component decay of the spin-echo signal. The single component decay character of the spin echo indicates water tightly bound by alpha-crystallin. Under a relative humidity P/P₀ = 1.0, collagen and BSA adsorbed, correspondingly, 19.3 and 28% of water and showed a two-component decay curve with T₂ about 5 and 40 msec. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with alpha-crystallin with similar distribution patterns in the lens layers. To conclude, it was found that alpha-crystallin can immobilize water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of alpha-crystallin and its superhydration properties and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.

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KEY WORDS: NMR spin-echo, bovine lens, chaperone, alpha-crystallin, isoelectric focusing, protein hydration, superhydration

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