Le Maire M, Thauvette L, de Foresta B, Viel A, Beauregard G, Potier M
Centre de Génétique Moléculaire, Université de Paris VI, Gif-sur-Yvette, France.
Biochem J. 1990 Apr 15;267(2):431-9. doi: 10.1042/bj2670431.
We have reinvestigated the use of ionizing radiations to measure the molecular mass of water-soluble or membrane proteins. The test was performed by using the most straightforward aspect of the technique, which consists of SDS/PAGE analysis of the protein-fragmentation process. We found that exposure of purified standard proteins to increasing doses of ionizing radiation causes progressive fragmentation of the native protein into defined peptide patterns. The coloured band corresponding to the intact protein was measured on the SDS gel as a function of dose to determine the dose (D37.t) corresponding to 37% of the initial amount of unfragmented protein deposited on the gel. This led to a calibration curve between 1/D37.t and the known molecular mass of the standard proteins whose best fit gave Mr = 1.77 x 10(6)/D37.t at -78 degrees C, i.e. 35% higher than the generally accepted value at that temperature obtained from inactivation studies. However, we have to conclude that this method is useless to determine the state of aggregation of a protein, since, for all the oligomers tested, the best fit was obtained by using the protomeric molecular mass, suggesting that there is no energy transfer between promoters. Furthermore, SDS greatly increases the fragmentation rate of proteins, which suggests additional calibration problems for membrane proteins in detergent or in the lipid bilayer. But the main drawback of the technique arises from our observation that some proteins behaved anomalously, leading to very large errors in the apparent target size as compared with true molecular mass (up to 100%). It is thus unreliable to apply the radiation method for absolute molecular-mass determination. We then focused on the novel finding that discrete fragmentation of proteins occurs at preferential sites, and this was studied in more detail with aspartate transcarbamylase. N-Terminal sequencing of several radiolysis fragments of the catalytic chain of the enzyme revealed that breaks along the polypeptide chains are localized close to the C-terminal end. Examination of the three-dimensional structure of aspartate transcarbamylase suggests that radiolysis sites (fragile bonds) might be localized in connecting loops.
我们重新研究了利用电离辐射来测定水溶性或膜蛋白分子量的方法。该测试是通过使用该技术最直接的方面来进行的，即对蛋白质片段化过程进行SDS/PAGE分析。我们发现，将纯化的标准蛋白暴露于递增剂量的电离辐射下会导致天然蛋白逐渐断裂成特定的肽段模式。在SDS凝胶上测量与完整蛋白相对应的有色条带随剂量的变化，以确定与凝胶上未断裂蛋白初始量的37%相对应的剂量（D37.t）。这得出了1/D37.t与标准蛋白已知分子量之间的校准曲线，在-78℃下其最佳拟合结果为Mr = 1.77 x 10(6)/D37.t，即比该温度下从失活研究中获得的普遍接受值高35%。然而，我们不得不得出结论，该方法对于确定蛋白质的聚集状态毫无用处，因为对于所有测试的寡聚体，通过使用原体分子量获得了最佳拟合，这表明启动子之间不存在能量转移。此外，SDS大大提高了蛋白质的断裂速率，这表明对于洗涤剂或脂质双层中的膜蛋白存在额外的校准问题。但该技术的主要缺点源于我们的观察，即一些蛋白质表现异常，导致与真实分子量相比，表观靶标大小出现非常大的误差（高达100%）。因此，应用辐射方法进行绝对分子量测定是不可靠的。然后我们关注到一个新发现，即蛋白质在优先位点发生离散断裂，并且用天冬氨酸转氨甲酰酶对此进行了更详细的研究。对该酶催化链的几个辐射分解片段进行N端测序表明，沿多肽链的断裂位于靠近C端的位置。对天冬氨酸转氨甲酰酶三维结构的研究表明，辐射分解位点（脆弱键）可能位于连接环中。
