Babizhayev Mark A, Nikolayev Gennady N, Goryachev Sergey N, Bours Johan
Moscow Helmholtz Research Institute of Eye Diseases, 14/19 Sadovaya Chernogryazskaya, Moscow, Russian Federation.
Biochim Biophys Acta. 2002 Jul 29;1598(1-2):46-54. doi: 10.1016/s0167-4838(02)00314-x.
The water-binding properties of bovine lens alpha-crystallin, collagen from calf skin and bovine serum albumin (BSA), were investigated with various techniques. The water absorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. 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, absorbed on the proteins. Isolated bovine lenses were sectioned into 11-12 morphological layers (from anterior cortex through nucleus to posterior cortex). Crystallin profiles were obtained for each lens layer using thin-layer isoelectric focusing in polyacrylamide gel (IEF). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During the water vapor uptake P/P(0)=0.75, alpha-crystallin did not absorb water, suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At P/P(0)=1.0, the absorption of water by alpha-crystallin was 17% with a single component decay character of spin-echo (T2=3 ms). Addition of water to alpha-crystallin to about 50% of its w/w in the protein sample showed T2=8 ms with only one single component decay of the spin-echo signal. The single component decay character of the spin-echo indicates at the tightly bound water by alpha-crystallin. Under a relative humidity P/P(0)=1.0, collagen and BSA absorbed correspondingly 19.3% and 28% of water and showed a two-component decay curve with T2 of about 5 and 40 ms. 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. 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. To conclude, it was found that alpha-crystallin can immobilize and bind 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 have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.
利用多种技术研究了牛晶状体α-晶体蛋白、小牛皮胶原蛋白和牛血清白蛋白（BSA）的水结合特性。在高真空解吸实验中通过体积法获得吸水能力，在可控气氛实验中通过重量法获得吸水能力。利用核磁共振自旋回波技术研究蛋白质样品的水合作用，并测定吸附在蛋白质上的水分子质子的自旋-自旋弛豫时间（T2）。将分离出的牛晶状体切成11 - 12个形态学层面（从前皮质穿过核到后皮质）。使用聚丙烯酰胺凝胶薄层等电聚焦（IEF）获得每个晶状体层面的晶体蛋白分布图。测定了各个晶状体形态学层面中蛋白质干重对应的含水量。在水蒸气吸收过程中，当P/P(0)=0.75时，α-晶体蛋白不吸水，这表明该蛋白质的疏水区域暴露于水性溶剂中。当P/P(0)=1.0时，α-晶体蛋白的吸水量为17%，自旋回波具有单组分衰减特征（T2 = 3毫秒）。向α-晶体蛋白中加入水，使其在蛋白质样品中的重量比达到约50%时，自旋回波信号仅呈现单组分衰减，T2 = 8毫秒。自旋回波的单组分衰减特征表明α-晶体蛋白对水的紧密结合。在相对湿度P/P(0)=1.0下，胶原蛋白和BSA的吸水量分别为19.3%和28%，并呈现出双组分衰减曲线，T2约为5毫秒和40毫秒。这些发现表明胶原蛋白和BSA中存在两种在空间上分离的水分级分。IEF数据表明水与α-晶体蛋白紧密结合，在晶状体各层面具有相似的分布模式。IEF数据表明α-晶体蛋白在晶状体的核和内皮质中可能具有类似伴侣蛋白的功能，但在外皮质中则不然。总之，发现α-晶体蛋白比其他蛋白质如胶原蛋白和BSA能在更大程度上固定和结合水。这些结果为α-晶体蛋白的结构特性提供了新的线索，对于理解该蛋白在晶状体和非眼组织中类似伴侣蛋白作用的机制具有重要意义。
