中华黄萤荧光素酶的性质

近年来,萤火虫荧光素酶催化的生物发光反应被广泛的应用到ATP检测、细菌数目检测、环境监测和其他生物学研究之中。通过对中华黄萤荧光素酶的性质研究,发现其分子量为6 400左右,是一种非典型的Michaelis-Menten氏酶,对底物之一的ATP动力学曲线为S型。最适反应温度为20～25℃,对温度极其敏感,当反应温度高于30℃时,该酶的活性快速失活。该荧光素酶的最适pH=6.5。随着pH值的下降该荧光素酶生物发光的最大发射光波长没有明显的红移现象。Mg²⁺和Mn²⁺对该酶活性的激活效果比较接近,最佳的激活浓度均在1mmol/L附近,而Ca²⁺最佳的激活浓度则小于1 mmol/L,且激活的效果只有Mg²⁺作用效果的50%左右。和Hg²⁺、Cd²⁺等离子一样,Cu²⁺会在一定程度上抑制该荧光素酶的活性。该酶的稳定性较差,对盐浓度和光照比较敏感,高浓度的盐类和长时间的日光照射能够使酶失活。谷胱甘肽(GSH)和二硫苏糖醇(DTT)是该酶较好的保护剂,可以提高该酶在一定时间内的稳定性,其中前者的作用效果要好于后者。

The Properties of Firefly Luciferase from Lucoila Chinesis L

Recent years,Bioluminescence which was catalyzed by firefly lueiferase involving lueiferin and ATP has been widely applied into the detection of ATP,counting of bacterium,environmental protection,new drugs screening and other biology researches.The firefly lueiferase in this research was extracted in Lucoila Chinesis L from ZheJiang province.Lucoila Chinesis L was a particular firefly which only inhabited in china mainland. First,Enzymology properties of firefly luciferase from Lucoila Chinesis L in this research was investigated that:Its relatively molecular mass was 6 400,Dynamic studies showed that it didn t behave characteristic of the typical Meehaelis-Menten enzyme.Both the dynamic curve for ATP and luciferin indicated an S shape. The optimum pH and temperature for the reaction involved the firefly luciferase were 6.5 and 20～25℃, respectively,and the activity of the firefly luciferase completely lost when the reaction temperature is above 30℃.This phenomenon indicated that the firefly luciferase was sensitive to the change of temperature. Second,some spectral characteristics also investigated.The wave length in the peak of light emission released from the reaction catalyzed by this firefly lueiferase did not demonstrated"red shift"phenomena when pH value dropped.Mg²⁺ and Mn²⁺ did the same thing when activating the firefly luciferase to a certain extent, while the Ca²⁺ did the 50% effect that Mg²⁺ or Mn²⁺ did.Like the Hg²⁺ and Cd²⁺,the Cu²⁺ can inhibit the activity of firefly luciferase in a certain degree.The firefly luciferase’s stability was weaker and it was very sen-sitive to the sun light and salt of high concentration.GSH and DTT were very useful in protecting the activity of the enzyme which can keep the activity of firefly luefferase during certain periods,and the GSH did a better job than the DTT did in protecting and enhencing the stability of this firefly luciferase under the same circum-stance.