卵黄高磷蛋白的分离与检测

Separation and Determination of the Phosvitin

蛋白质的可逆磷酸化作用在细胞间的信息传输中非常重要。蛋白质中所含磷氨酸最常见的是磷氨基丝氨酸(P-Ser),磷苏氨酸(P-Tyr)也有发现,只是不常见。检测以氨基酸形式存在的磷氨酸,对理解掌握许多蛋白质的作用非常重要,有一些还与信息通道有关。本文论证了以带电氨基酸形式存在的磷氨酸的分离,如天门冬氨酸(Asp)和谷氨酸,通过间接检测磷酸腺苷(AMP),可以利用毛细管电泳进行有效的分离。在没有天门冬氨酸(Asp)、谷氨酸或磷酸盐残渣的干扰下,磷酪氨酸,磷氨基丝氨酸和磷苏氨酸的基线溶液在检测极限为磷氨酸1mg/L的情况下,经大约18min就可检测出来。该法已用于含磷氨基丝氨酸的卵黄高磷蛋白的酸水解过程,残渣量与钼酸盐比色直接相关。

The reversible phosphorylation of proteins is important in intercellular communication. The most frequentlyphosphorylated amino acid in proteins is phosphoserine (P-Ser);however, phosphothreonine(P-thr),phosphotyrosine (P-Tyr).And phosphoarginine(P-Arg) are also found in decreasing frequency. The derection of phosphorylated amino acids in thepresence of amino acids is important to an understanding of the role of many proteins, some of which are involved in signaltransduction pathways.We have shown that separation of these phosphoamino acids in the presence of similarly charged amino acids,such as asparticacid(Asp) and glutamic acid (Glu) can be effectively done by capillary electrophoresis (CE) with indirect detection usingadenosine monophosphate(AMP). Baseline resolution of P-Tyr,P-Ser, without interference of Asp and Glu or residual phosphate,in about 18 min with detection limits of 1 mg/L(3～6μm) for the phosphoamino acids is possible.This CE method has been usedto follow the acids hydrolysis of phosvitin,a P-Ser containing protein, and the number of residues found correlates statisticallyto the molybdate colorimetric method.