Wood K V
Department of Chemistry, University of California, San Diego, La Jolla 92093.
J Biolumin Chemilumin. 1990 Apr-Jun;5(2):107-14. doi: 10.1002/bio.1170050206.
Luminescence assays are generally based on measurements of light intensity alone. Inclusion of colour as an additional parameter of the assay could increase the information content. Colour variation in luminescence is particularly prevalent among beetle luciferases. To study the relationship between enzyme structure and colour, luciferases from a Jamalcan click beetle were examined as a model system. These luciferases emit light ranging from green to orange, though their amino acid sequences differ by less than 5%. Through mutation of their respective cDNA clones, the amino acids responsible for the colour variation were identified. These specific amino acids are few, and they act upon colour independently with respect to the enzyme structure. Analysis of their effects indicates that the potential for colour variation among beetle luciferases is greater than is evident among the click beetle luciferase. Because of the subtle changes of enzyme structure that effect colour, luciferases that emit different colours may be useful as paired genetic reporters. They should interact equivalently with the intracellular environment of a host, but could be distinguished by colour in their assay. Such paired reporters could be used to observed simultaneous events, or to provide internal control for luminescence measurements.
发光测定通常仅基于光强度的测量。将颜色作为测定的一个附加参数纳入可以增加信息含量。发光中的颜色变化在甲虫荧光素酶中尤为普遍。为了研究酶结构与颜色之间的关系，以一种牙买加叩头虫的荧光素酶作为模型系统进行了研究。这些荧光素酶发出从绿色到橙色的光，尽管它们的氨基酸序列差异小于5%。通过对它们各自的cDNA克隆进行突变，确定了导致颜色变化的氨基酸。这些特定的氨基酸数量很少，并且它们相对于酶结构独立地作用于颜色。对它们作用的分析表明，甲虫荧光素酶之间颜色变化的潜力大于叩头虫荧光素酶中明显的情况。由于影响颜色的酶结构的细微变化，发出不同颜色光的荧光素酶可能作为配对的遗传报告物有用。它们应该与宿主的细胞内环境等效相互作用，但在测定中可以通过颜色区分。这种配对的报告物可用于观察同时发生的事件，或为发光测量提供内部对照。
