Association between lumazine protein and bacterial luciferase: direct demonstration from the decay of the lumazine emission anisotropy.
Association between lumazine protein and bacterial luciferase: direct demonstration from the decay of the lumazine emission anisotropy.
复制标题
二氢吲嗪蛋白与细菌荧光素酶之间的关联：二氢吲嗪发射各向异性衰减的直接证明。
作者：
Visser,AJ;Lee,J
安东尼G. Visser和John Lee* 摘要：发光杆菌发光氨嗪蛋白中结合的6，7-二甲基-8-核糖基发光氨嗪的发射各向异性衰减的相关时间为23 ns（3 ℃）。在存在约100 µ浓度的从哈氏弧菌纯化的细菌荧光素酶的情况下，其摩尔过量约为鲁马嗪蛋白的10倍，相关时间增加至约75 ns，并且各向异性以单指数衰减。这是荧光素酶和Lumazine蛋白之间关联的直接证明，并且这通过沉降速度测量得到证实。单独的Lumazine蛋白质的沉降系数（s_（20）w = 1-9 S）在Lu-ciferase溶液中提高到5.6S。在较低的荧光素酶浓度的各向异性decaybecomes双指数，和一个受约束的相关时间分析用于提取振幅的较短和较长的时间作为措施的自由和复杂的鲁马嗪蛋白浓度。使用该技术进行分析，用鲁马嗪蛋白滴定显示，在发光杆菌属的常规体外生物发光细菌中具有高活性的脱氢酶以高产率产生鲁马嗪蛋白（Gast & Lee，1978; Lee &科卡，1978; Lee & Elrod，1981）。这种20000道尔顿的可溶性蛋白质被称为lumazine蛋白，因为它含有6，7-二甲基-8-核糖基lumazine（科卡和李，1979）（下文称为“lumazine”）作为结合辅基（Kd= 0.05 μ，2 C;维瑟和李，1980;欧文等人，1980年）。结合的鲁马嗪是有效的荧光（gF*= 0.6，tf= 14.4
Antonie J. W. G. Visser and John Lee* abstract: The correlation time for the decay of the emission anisotropy of the bound 6, 7-dimethyl-8-ribityllumazine in the lumazine protein from Photobacterium phosphoreum is 23 ns (3 C). In the presence of about 100 µ concentrations of the bacterial luciferase purified from Vibrio harveyi, in about 10 times molar excess over the lumazine protein, the correlation time is increased to around 75 ns, and the an-isotropy decays as a single exponential. This is a direct dem-onstration of association between luciferase and lumazine protein, and this is confirmed by sedimentation velocity measurements. The sedimentationcoefficient for lumazine protein alone, s20w= 1-9 S, is increased to 5.6 S in the lu-ciferase solution. At lower luciferase concentrations the an-isotropy decaybecomes biexponential, and a constrained correlation times analysis is used to extract amplitudes of the shorter and longer times as measures of the free and complexed lumazine protein concentrations. With the use of this technique for analysis, a titration with lumazine protein shows that luciferases, which have high activities in the regular in vitroBioluminescent bacteria of the genus Photobacterium produce lumazine protein in high yield (Gast & Lee, 1978; Lee & Koka, 1978; Lee & Elrod, 1981). This soluble protein of 20 000 daltons is called lumazine protein because it contains 6, 7-dimethyl-8-ribityllumazine (Koka & Lee, 1979)(here-inafter called “lumazine”) as a bound prosthetic group (Kd= 0.05 µ, 2 C; Visser & Lee, 1980; Irwin et al., 1980). The bound lumazine is efficiently fluorescent (gF*= 0.6, tf= 14.4