Megli F M, De Lisi A, Quagliariello E
Dipartimento di Biochimica e Biologia Molecolare, Università di Bari, Italy.
Anal Biochem. 1990 Aug 1;188(2):390-3. doi: 10.1016/0003-2697(90)90625-j.
Bovine lung annexins p32 and p34 were spin labeled with an iodoacetamidoproxyl spin label, a reagent that reportedly couples with protein methionine residues. Labeling conditions and stoichiometry were studied with the radiolabeled analogue [1-14C]iodoacetamide. As judged by this method, carboxamidomethylation of both p32 and p34 occurred up to a 0.7 mol ratio after 60 h of reaction at 37 degrees C and at pH 4. The two proteins retained Ca2(+)-dependent phospholipid-binding ability both in radiolabeled and in spin-labeled forms. Electron resonance spectra of spin-labeled p32 and p34 showed the features of a partially immobilized spin probe, with rotational correlation time values of 1.15 and 1.25 ns, respectively, which definitely indicate successful spin labeling. Quantitation of ESR spectra by computer double integration indicated 70% spin labeling of both proteins, as anticipated by radiolabeling. The use of spin-labeled p32 and p34 in the study of Ca2(+)-dependent interaction of annexins with biomembranes is proposed.
牛肺膜联蛋白p32和p34用碘乙酰胺基氧基自旋标记物进行自旋标记，该试剂据报道可与蛋白质甲硫氨酸残基结合。用放射性标记类似物[1-14C]碘乙酰胺研究标记条件和化学计量。用这种方法判断，在37℃和pH 4下反应60小时后，p32和p34的羧酰胺甲基化反应达到0.7摩尔比。这两种蛋白质在放射性标记和自旋标记形式下均保留了Ca2(+)依赖性磷脂结合能力。自旋标记的p32和p34的电子共振光谱显示了部分固定化自旋探针的特征，旋转相关时间值分别为1.15和1.25纳秒，这明确表明自旋标记成功。通过计算机双积分对电子自旋共振光谱进行定量分析表明，两种蛋白质的自旋标记率均为70%，这与放射性标记预期的结果一致。本文提出将自旋标记的p32和p34用于研究膜联蛋白与生物膜的Ca2(+)依赖性相互作用。
