Muramoto K, Nokihara K, Ueda A, Kamiya H
School of Fisheries Sciences, Kitasato University, Iwate, Japan.
Biosci Biotechnol Biochem. 1994 Feb;58(2):300-4. doi: 10.1271/bbb.58.300.
A fluorescent Edman-type reagent, fluorescein isothiocyanate (FITC), was adapted to a gas-phase sequencer fitted with a miniature reaction chamber. Fluorescein thiohydantion amino acids were identified by on-line gradient HPLC with fluorescence monitoring. The optimized protocol for the coupling reaction using FITC and phenylisothiocyanate, and the degree of washing to remove the excess reagents were established. Myoglobin (5 pmol) and lysozyme (5 pmol) were analyzed by the sequencer to obtain a 35-51% initial yield (I.Y.) and 82-88% repetitive yield (R.Y.) for the former, and 51-66% I.Y. and 91-92% R.Y. for the latter. These figures permitted 20-25 amino acid residues to be identified from the N-terminus of 5 pmol samples applied to the sequencer.
一种荧光埃德曼型试剂，异硫氰酸荧光素（FITC），被应用于配备微型反应室的气相测序仪。通过在线梯度高效液相色谱结合荧光监测来鉴定硫代乙内酰脲荧光素氨基酸。确定了使用FITC和异硫氰酸苯酯进行偶联反应的优化方案，以及去除过量试剂的洗涤程度。用该测序仪分析了肌红蛋白（5皮摩尔）和溶菌酶（5皮摩尔），前者的初始产率（I.Y.）为35 - 51%，重复产率（R.Y.）为82 - 88%；后者的初始产率为51 - 66%，重复产率为91 - 92%。这些数据使得能够从应用于测序仪的5皮摩尔样品的N端鉴定出20 - 25个氨基酸残基。
