Garcia-Barreno P, Guisasola M C, Suarez A
Unidad de Medicina y Cirugia Experimental, Hospital General, Universitario Gregorio Marañón, Doctor Esquerdo, 46, 28007, Madrid, Spain.
Comp Biochem Physiol B Biochem Mol Biol. 2005 Jun;141(2):179-85. doi: 10.1016/j.cbpc.2005.02.012.
Water soluble proteins (WSPs) in Sus scrofa lenses from pigs in different developmental stages: (young (GI), young adult (GII), and middle-aged (GIII)) were separated using GF-HPLC, yielding fractions of different molecular weights. Non-tryptophan (345/420 nm) and tryptophan (280/345 nm) fluorescence was measured in these fractions. Relative non-tryptophan fluorescence increased with age at a rate directly correlated to the molecular weight of aggregates forming the different chromatographic fractions, while tryptophan fluorescence tended to decrease. The crystallins constituting each fraction were separated using 2D-electrophoresis and after development with Coomassie blue they were identified using MS-TOF. Also, the protein content of each spot was quantified by subsequent scanning and integration. The proportions of unchanged crystallins characteristically changed with age in chromatographic fractions of different molecular weights. Thus it was possible to relate these changes with those occurring in the fluorescent properties and molecular weight of supramolecular structures.
不同发育阶段猪（幼年（GI）、青年成年（GII）和中年（GIII））的猪晶状体中的水溶性蛋白质（WSPs）通过凝胶过滤高效液相色谱（GF-HPLC）进行分离，得到不同分子量的组分。对这些组分测量了非色氨酸（345/420 nm）和色氨酸（280/345 nm）荧光。相对非色氨酸荧光随年龄增加，其速率与形成不同色谱组分的聚集体的分子量直接相关，而色氨酸荧光则趋于下降。使用二维电泳分离构成每个组分的晶状体蛋白，用考马斯亮蓝染色后，使用基质辅助激光解吸电离飞行时间质谱（MS-TOF）进行鉴定。此外，通过随后的扫描和积分对每个斑点的蛋白质含量进行定量。不同分子量色谱组分中特征性不变的晶状体蛋白比例随年龄变化。因此，有可能将这些变化与超分子结构的荧光特性和分子量变化联系起来。
