Charbonneau H, Cormier M J
J Biol Chem. 1979 Feb 10;254(3):769-80.
A Ca2+-triggered luciferin-binding protein (BP-LH2) from the bioluminescent marine coelenterate, Renilla reniformis, has been purified by conventional methods. One kilogram of processed animals yields approximately 2.7 mg of pure protein with an overall yield of 55%. Physicochemical studies show that BP-LH2 is a globular protein containing one single polypeptide chain with one disulfide bond. Ultracentrifugation studies, amino acid analysis, and sodium dodecyl sulfate-gel electrophoresis show that BP-LH2 has an average molecular weight of 18,500. BP-LH2 has a Stokes radius of 23 A, a sedimentation coefficient, S020,w, of 2.3 S, and an isoelectric point of 4.3. The acidic nature of the protein was confirmed by amino acid analysis, which showed that 27% of the residues are acidic. The protein contains no carbohydrate, phosphate, or tryptophan. There is one noncovalently bound molecule of coelenterate type luciferin resulting in distinct protein spectral properties with absorption maxima at 276 nm (epsilon 0.1% 276 = 1.31) and 446 nm (episoln 0.1% 446 = 0.47) and a fluorescence emission at 520 nm (uncorrected). In the presence of Ca2+, BP-LH2 will react with Renilla luciferase to give the characteristic in vitro blue bioluminescence. Ca2+ binding produces a distinct change in the spectral properties of BP-LH2 including a 4-fold enhancement of tyrosine fluorescence at 332 nm and a 5-fold fluorescence enhancement at 520 nm. In addition, the visible absorption maximum shifts from 446 nm to 420 nm. The fluorescence enhancement at 320 nm occurs over the range from 1 to 10 micrometer Ca2+. BP-LH2 has two Ca2+-binding sites with an estimated Kd of 0.02 micrometer, in 10 muM Tris at pH 7.2. BP-LH2 was compared to several well studied Ca2+-binding proteins and was found to possess similar Ca2+-binding and physicochemical properties. This study clearly demonstrates that BP-LH2 is capable of triggering a bioluminescent flash in response to an intracellular Ca2+ transient.
一种从发光海洋腔肠动物海肾（Renilla reniformis）中提取的钙离子触发型荧光素结合蛋白（BP-LH2）已通过传统方法纯化。一千克经过处理的动物可产出约2.7毫克纯蛋白，总产率为55%。物理化学研究表明，BP-LH2是一种球状蛋白，含有一条单多肽链和一个二硫键。超速离心研究、氨基酸分析和十二烷基硫酸钠-凝胶电泳表明，BP-LH2的平均分子量为18,500。BP-LH2的斯托克斯半径为23埃，沉降系数S020,w为2.3 S，等电点为4.3。氨基酸分析证实了该蛋白的酸性性质，结果显示27%的残基为酸性。该蛋白不含碳水化合物、磷酸盐或色氨酸。存在一个非共价结合的腔肠动物型荧光素分子，导致该蛋白具有独特的光谱特性，吸收最大值分别在276纳米（ε0.1% 276 = 1.31）和446纳米（ε0.1% 446 = 0.47），荧光发射峰在520纳米（未校正）。在钙离子存在的情况下，BP-LH2会与海肾荧光素酶反应，产生特征性的体外蓝色生物发光。钙离子结合会使BP-LH2的光谱特性发生明显变化，包括332纳米处酪氨酸荧光增强4倍，520纳米处荧光增强5倍。此外，可见吸收最大值从446纳米移至420纳米。320纳米处的荧光增强发生在1至10微摩尔钙离子浓度范围内。在pH 7.2的10微摩尔 Tris缓冲液中，BP-LH2有两个钙离子结合位点，估计解离常数Kd为0.02微摩尔。将BP-LH2与几种经过充分研究的钙离子结合蛋白进行比较，发现它具有相似的钙离子结合和物理化学性质。这项研究清楚地表明，BP-LH2能够响应细胞内钙离子瞬变触发生物发光闪光。
