Fukuoka Industrial Technology Center, 1465-5 Aikawa, Kurume, Fukuoka, 839-0861, Japan,
Cytotechnology. 2005 Jan;47(1-3):59-67. doi: 10.1007/s10616-005-3757-4.
Proteins of plasma membrane could be an index of purification of the plasma membrane of animal cells. A convenient method is proposed for determining the plasma membrane proteins by a surface plasmon resonance (SPR) biosensor. Biotinylated proteins were observed only in the peripheral areas of MOLT-4 cells which were treated by 5-[5-(N-succinimidyloxycarbonyl) pentylamido] hexyl-D: -biotinamide. The proteins on HeLa cells were also biotinylated. And then the membrane samples of the HeLa cells were injected onto the avidin-immobilized SPR-surface, and components bound non-specifically on the surface were removed by a washout solution. The amount of biotinylated protein (BP) was determined directly from the absolute resonance unit (RU) after injection of the washout solution. In the method a reference surface was not needed. The amount of BP bound to the surface was gradually attenuated with the repeated injection, and a method for calibrating the RU value was introduced by considering the ratio of attenuation by every injection. The correlation between the BP titer calculated by the calibration and the theoretically-estimated one was greatly improved. Three cycles of the BP determination on a sensor surface was performed successfully. During the purification process of membrane fractions, the degree of purification as judged by the BP titer was in good agreement with the degree of increase in aminopeptidase N activity in the membrane fraction. Thus, the BP titer could be used as an index for purification of plasma membrane.
质膜蛋白可以作为动物细胞质膜纯化的指标。本文提出了一种利用表面等离子体共振（SPR）生物传感器测定质膜蛋白的简便方法。用 5-[5-(N-琥珀酰亚胺基氧基羰基)戊基酰胺基]己基-D：-生物素酰胺处理 MOLT-4 细胞后，只有在细胞的外周区域观察到生物素化蛋白。HeLa 细胞的蛋白也被生物素化。然后将 HeLa 细胞膜样品注入到亲和素固定的 SPR 表面，并用洗脱液去除非特异性结合在表面上的成分。在注入洗脱液后，直接从绝对共振单位（RU）中确定生物素化蛋白（BP）的量。该方法不需要参考表面。随着重复注入，BP 结合到表面的量逐渐衰减，通过考虑每次注入的衰减比，引入了一种校准 RU 值的方法。通过校准计算的 BP 效价与理论估计值之间的相关性得到了很大改善。在传感器表面上成功地进行了三次 BP 测定循环。在膜部分的纯化过程中，根据 BP 效价判断的纯化程度与膜部分中氨肽酶 N 活性的增加程度非常吻合。因此，BP 效价可作为质膜纯化的指标。
