Probing different conformational states of bovine alpha-lactalbumin: fluorescence studies with 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate].
Probing different conformational states of bovine alpha-lactalbumin: fluorescence studies with 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate].
复制标题
探索牛 α-乳清蛋白的不同构象状态：4,4-双[1-(苯基氨基)-8-萘磺酸盐]的荧光研究。
DOI：
10.1021/bi00336a006
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Berliner,LJ
中科院分区：
文献类型：
--
作者：
Musci,G;Berliner,LJ
材料与方法蛋白质.牛-LA来自Sigma Chemical Co. (lot 52 F-8075 - 1），其通常含有0.34 mol Ca（II）/mol蛋白质（未处理的Sigma-LA）。载脂蛋白形式是通过反复使蛋白质通过三（羧甲基）乙二胺柱来制备的。根据原子吸收和NMR（K。古贺和L. J. Berliner，未发表的结果）。化学品Bis-ANS（4，4，-双[1-（苯基氨基）-8-萘磺酸]二钾盐）购自Mo-lecular Probes，联合城，OR。其浓度由在385 nm处的光吸收估计，t = 16790 M-1cm-1（Farris等人，1978年）。超纯氯化锰（99.999%，批号0518）和氯化锌（99.999%，批号0208）来自Aldrich Chemical Co.超纯EDTA（99 + %，批号011581）来自Alpha Products。所有其他试剂均为分析级，无需进一步纯化即可使用。方法.在SLM 4800S型和Perkin-Elmer MPF44A型分光荧光计上在25 ℃下进行荧光测量。如前所述，通过非线性回归分析拟合平衡结合数据（Murakami等人，1982年）。通过280 nm处的光吸收估计蛋白质浓度（e = 2.01 mg-1 mL-1）。在Perkin-Elmer Model 360上进行原子吸收测量。
Materials and MethodsProteins. Bovine-LA was from Sigma Chemical Co.(lot 52F-8075-1), which typically contained 0.34 mol of Ca (II)/mol of protein (untreated Sigma-LA). The apo form was prepared by repeatedly passing the protein down a column of tris (carboxymethyl) ethylenediamine. It contained less than 2% bound calcium, as estimated from atomic absorption and NMR (K. Koga and L. J. Berliner, unpublished results). Chemicals. Bis-ANS (4, 4,-bis [l-(phenylamino)-8-naphthalenesulfonic acid] dipotassium salt) was from Mo-lecular Probes, Junction City, OR. Its concentration was estimated from the optical absorption at 385 nm, t= 16 790 M" 1 cm-1 (Farris et al., 1978). Ultrapure manganese chloride (99.999%, lot 0518) and zinc chloride (99.999%, lot 0208) were from Aldrich Chemical Co. Ultrapure EDTA (99+%, lot 011581) was from Alpha Products. All other reagents were of analytical grade and were used without further purification. Methods. Fluorescence measurements were carried out on SLM Model 4800S and Perkin-Elmer Model MPF44A spectrofluorometers at 25 C. Equilibrium binding data were fit by nonlinear regression analysis as noted earlier (Murakami et al., 1982). Protein concentration was estimated by optical absorption at 280 nm (e= 2.01 mg" 1 mL" 1). Atomic ab-sorption measurements were carried out on a Perkin-Elmer Model 360.