Burleigh B D, Liu W K, Ward D N
J Biol Chem. 1976 Jan 25;251(2):308-15.
Tetranitromethane reaction with intact ovine lutropin and its isolated subunits was studied using spectrophotometric measurements, amino acid analysis, and isolation of tyrosyl peptides. Tyrosyl residues in the beta subunit (beta37, beta59) did not react with tetranitromethane in the intact hormone, but were nitrated in the isolated subunit. The sequence and extent of reaction of tetranitromethane with the tyrosyl residues in the alpha subunit was alpha21 = alpha92 = alpha93 (in intact hormone or isolated subunit) greater than alpha 41 (reacted in isolated subunit only) greater than alpha 30 (reacted in isolated subunit in 8 M urea only). Polymerization was observed as a side reaction in agreement with previous studies. The degree of polymerization appeared to be related to both primary sequence and tertiary structure, and for lutropin had the relation: alpha subunit (93% polymerized) greater than intact hormone greater than beta subunit (less than 40%). Polymerization observed with vasopressin was significantly greater than with oxytocin; for these peptides the tyrosine residues in the monomeric product were converted to 3-nitrotyrosine. Neither 3-nitrotyrosine nor tyrosine was detected in the polymerized by-products. In the tetranitromethane reaction with intact ovine lutropin, other reaction products charcterized by absorption spectra were found. Peptides isolated from these products lacked the characteristic 428 nm abosrption maxima of 3-nitrotyrosyl peptides and showed instead absorption in the 310 to 350 nm region. Similar products from tetranitromethane reactions with di- and tripeptides containing tyrosine have been observed previously (Boyd, N.D., and Smith, D.B. (1971) Can. J. Biochem, 49, 154-161), but they have not been studied in proteins. A possible relationship to the polymerization side reaction is suggested.
利用分光光度测量、氨基酸分析以及酪氨酰肽的分离，研究了四硝基甲烷与完整的绵羊促黄体激素及其分离亚基的反应。在完整激素中，β亚基中的酪氨酰残基（β37、β59）不与四硝基甲烷反应，但在分离的亚基中被硝化。四硝基甲烷与α亚基中酪氨酰残基的反应顺序和程度为α21 = α92 = α93（在完整激素或分离亚基中）大于α41（仅在分离亚基中反应）大于α30（仅在8M尿素中的分离亚基中反应）。与先前的研究一致，观察到聚合反应是一种副反应。聚合程度似乎与一级序列和三级结构都有关，对于促黄体激素，其关系为：α亚基（93%聚合）大于完整激素大于β亚基（小于40%）。观察到加压素的聚合反应明显大于催产素；对于这些肽，单体产物中的酪氨酸残基转化为3-硝基酪氨酸。在聚合副产物中未检测到3-硝基酪氨酸和酪氨酸。在四硝基甲烷与完整绵羊促黄体激素的反应中，发现了以吸收光谱为特征的其他反应产物。从这些产物中分离出的肽缺乏3-硝基酪氨酰肽特有的428nm吸收最大值，而是在310至350nm区域显示吸收。先前已观察到四硝基甲烷与含酪氨酸的二肽和三肽反应产生的类似产物（Boyd，N.D.，和Smith，D.B.（1971）Can.J.Biochem，49，154 - 161），但尚未在蛋白质中进行研究。提示了与聚合副反应的可能关系。
