PRECISION OF SODIUM DODECYL SULFATE-POLYACRYLAMIDE-GEL ELECTROPHORESIS FOR MOLECULAR-WEIGHT ESTIMATION OF A MEMBRANE GLYCOPROTEIN - STUDIES ON BOVINE RHODOPSIN
PRECISION OF SODIUM DODECYL SULFATE-POLYACRYLAMIDE-GEL ELECTROPHORESIS FOR MOLECULAR-WEIGHT ESTIMATION OF A MEMBRANE GLYCOPROTEIN - STUDIES ON BOVINE RHODOPSIN
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DOI：
10.1016/0003-9861(75)90001-6
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发表时间：
1975-01-01
影响因子：
3.9
通讯作者：
RODBARD, D
中科院分区：
文献类型：
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作者：
FRANK, RN;RODBARD, D
采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定蛋白质分子量的方法的精密度和准确度的评估标准已应用于牛视细胞外段膜的视紫红质。制备这种疏水蛋白用于电泳的各种方法在其溶解和解聚外节膜的多肽组分的能力上不同，从而导致蛋白条带的模式和视紫红质的表观分子量的变化。即使有最佳的增溶和解聚，视紫红质相对于一系列标准蛋白质的行为是这样的，随着丙烯酰胺浓度从4%增加到10%，表观分子量从40，400系统地降低到34，500。正如Ferguson曲线的logRf与凝胶浓度和分裂凝胶实验所证明的，这种差异是由以下事实解释的：视紫红质的零凝胶浓度（Y 0）的外推Rf显著低于用作分子量标准的可溶性蛋白质的Y 0。在这种情况下，可能更可靠的分子量估计是通过绘制延迟系数（KR）与分子量。如果只考虑Rfare的测量误差，并采用KR与分子量的二次关系，则牛视紫红质的测定值为29，500 ± 1000。使用KRvs分子量的加权线性回归，当考虑校准曲线中的不确定度时，我们获得的分子量估计值为32，700 ± 5000。由于与可溶性蛋白标准品相比，视紫红质的去污剂结合特性及其斯托克斯半径与其分子量的关系存在不确定性，因此必须谨慎看待这些值。
Criteria for assessing the precision and accuracy of methods for estimation of molecular weight for proteins using sodium dodecyl sulfate-polyacrylamide-gel electrophoresis have been applied to rhodopsin from bovine visual cell outer segment membranes. Various methods of preparing this hydrophobic protein for electrophoresis differ in their ability to solubilize and disaggregate polypeptide constituents of the outer segment membrane, with resultant variations in the pattern of protein bands and the apparent molecular weight of rhodopsin. Even with optimal solubilization and disaggregation, the behavior of rhodopsin relative to a series of standard proteins is such that the apparent molecular weight decreases systematically from 40,400 to 34,500 as the acrylamide concentration increases from 4 to 10%. As demonstrated by Ferguson plots of logRfvs gel concentration and split gel experiments, this discrepancy is explained by the fact that the extrapolatedRffor zero gel concentration (Y0) for rhodopsin is significantly lower than theY0's for the soluble proteins used as molecular weight standards. In such cases, a possibly more reliable molecular weight estimate is obtained by plotting the retardation coefficient (KR) vs molecular weight. This method yields a value of 29,500 ± 1000 for bovine rhodopsin if only the errors in measurement ofRfare considered and a quadratic relationship betweenKRand molecular weight is used. Using weighted linear regression forKRvs molecular weight, we obtain a molecular weight estimate of 32,700 ± 5000 when the uncertainty in the calibration curve is considered. Because of uncertainties regarding the detergent-binding properties of rhodopsin and the relationship of its Stokes radius to its molecular weight by comparison with the soluble protein standards, these values must be viewed with caution.