Mitsuhashi Tomiko, Kasai Midori, Hatae Keiko
Departmnent of Food and Nutrition, Junior College Nihon University, 2-31-145, Bunkyo cho, Mishima, Shizuoka 411-8555, Japan.
J Agric Food Chem. 2002 Dec 18;50(26):7499-503. doi: 10.1021/jf020362l.
An improved method was investigated for sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE) to facilitate the analysis of the giant myofibrillar proteins, connectin and nebulin, in fish meat by using jack mackerel (Trachurus japonicus) as the sample fish. It was established that separation of the alpha-connectin band from the beta-connectin band by SDS-PAGE could be achieved by using 3-5% gradient gels with glycerol to facilitate the formation of a gradient with polymerization at 35 degrees C. SDS-PAGE samples of white dorsal muscle from the jack mackerel were homogenized with a 2% SDS solution containing an inhibitor mixture (1 microg/mL of phenylmethanesulfonyl fluoride, 1 microg/mL of leupeptin, and 1 microg/mL of E-64) and heated at 50 degrees C for 20 min. Heating these samples at 100 degrees C for 2 min resulted in the disintegration of connectin but did not affect nebulin. A purified myofibril sample and a whole muscle sample showed similar changes in the overall rate of degradation of whole connectin and nebulin during the postmortem storage period, but it was clear that beta-connectin was cleaved from alpha-connectin during the preparation of myofibrils at the early stage postmortem. Storage of the SDS-PAGE samples at -85 degrees C was preferable to storage at -18 degrees C for a long period.
研究了一种改进的十二烷基硫酸钠聚丙烯酰胺平板凝胶电泳（SDS-PAGE）方法，以促进对鱼肉中巨大肌原纤维蛋白肌联蛋白和伴肌动蛋白的分析，使用日本竹荚鱼（Trachurus japonicus）作为样本鱼。结果表明，通过使用含甘油的3-5%梯度凝胶，在35℃聚合以促进梯度形成，可通过SDS-PAGE实现α-肌联蛋白带与β-肌联蛋白带的分离。将日本竹荚鱼白色背部肌肉的SDS-PAGE样品用含有抑制剂混合物（1μg/mL苯甲基磺酰氟、1μg/mL亮抑酶肽和1μg/mL E-64）的2% SDS溶液匀浆，并在50℃加热20分钟。在100℃加热这些样品2分钟会导致肌联蛋白分解，但不影响伴肌动蛋白。纯化的肌原纤维样品和全肌肉样品在死后储存期间，整个肌联蛋白和伴肌动蛋白的总体降解速率显示出相似的变化，但很明显，在死后早期制备肌原纤维的过程中，β-肌联蛋白从α-肌联蛋白上被切割下来。SDS-PAGE样品在-85℃储存比在-18℃长期储存更可取。
