Siezen R J, Bindels J G, Hoenders H J
Eur J Biochem. 1978 Nov 15;91(2):387-96. doi: 10.1111/j.1432-1033.1978.tb12691.x.
Cortial alpha-crystallin was size-fractionated by gel filtration on Ultrogel AcA22 and charge-fractionated by anion-exchange chromatography on DE-52 DEAE-cellulose using gradient elution. Electron microscopy demonstrates that both native and reassociated alpha-crystallin are heterogeneous populations of spherical or slightly ellipsoidal molecules with diameters of 13.5--16.0 nm (maximum at 14.0--15.0 nm) for native alpha-crystallin and 8.5--12.5 nm (maximum at 10.0--10.5 nm) for reassociated alpha-crystallin. An enormous charge heterogeneity of native alpha-crystallin was detected, which is shown to arise from variations in the stoichiometry of the 5 main types of subunits. The molar ratio of acidic chains (A2, A1 and A1/2-151) to basic chains (B2 and B1) varies from 70/30--80/20 (averaging about 3/1) and the amount of deamidated chains (A1 and B1) varies from 7--37%. Recombination of the subunits, after dissociation in 6 M urea, leads to a charge heterogeneity of reassociated alpha-crystallin very similar to that of native alpha-crystallin. Therefore, specific formation of pure A or B chain aggregates is not preferred. Instead, random combination of subunits is theoretically shown to be sufficient to describe the observed charge microheterogeneity of both reassociated and native alpha-crystallin. No obvious relationship exists between size and charge heterogeneity. Within these ranges of molecular weight and subunit composition there are more than 1000 different combinations of A2, A1, A1/2-151, B2 and B1 conceivable.
通过在Ultrogel AcA22上进行凝胶过滤对皮质α-晶状体蛋白进行尺寸分级，并使用梯度洗脱在DE-52 DEAE-纤维素上通过阴离子交换色谱进行电荷分级。电子显微镜显示，天然和重新缔合的α-晶状体蛋白都是球形或略呈椭圆形分子的异质群体，天然α-晶状体蛋白的直径为13.5 - 16.0 nm（最大值在14.0 - 15.0 nm），重新缔合的α-晶状体蛋白的直径为8.5 - 12.5 nm（最大值在10.0 - 10.5 nm）。检测到天然α-晶状体蛋白存在巨大的电荷异质性，这表明其源于5种主要亚基化学计量的变化。酸性链（A2、A1和A1/2 - 151）与碱性链（B2和B1）的摩尔比在70/30 - 80/20之间变化（平均约为3/1），脱酰胺链（A1和B1）的量在7 - 37%之间变化。在6 M尿素中解离后亚基的重组导致重新缔合的α-晶状体蛋白的电荷异质性与天然α-晶状体蛋白非常相似。因此，不倾向于特定形成纯A链或B链聚集体。相反，理论上表明亚基的随机组合足以描述观察到的重新缔合和天然α-晶状体蛋白的电荷微异质性。尺寸和电荷异质性之间不存在明显关系。在这些分子量和亚基组成范围内，可以设想A2、A1、A1/2 - 151、B2和B1有超过1000种不同的组合。
