EXTRACTION, PURIFICATION AND PROPERTIES OF AEQUORIN, A BIOLUMINESCENT PROTEIN FROM LUMINOUS HYDROMEDUSAN, AEQUOREA
EXTRACTION, PURIFICATION AND PROPERTIES OF AEQUORIN, A BIOLUMINESCENT PROTEIN FROM LUMINOUS HYDROMEDUSAN, AEQUOREA
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DOI：
10.1002/jcp.1030590302
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发表时间：
1962-01-01
影响因子：
--
通讯作者：
SAIGA, Y
中科院分区：
文献类型：
--
作者：
SHIMOMURA, O;JOHNSON, FH;SAIGA, Y
用EDTA提取，通过硫酸铵沉淀和DEAE柱层析纯化，得到一种具有蛋白质性质和在加入Ca++时具有高发光活性的物质，水母发光蛋白。除了Sr++的可疑结果外，未发现其他阳离子替代Ca++。通过透析、硫酸铵沉淀、DEAE层析或纸电泳，不能从水母发光蛋白中分离出发光活性组分。水母发光蛋白的紫外吸收在280 m[mu]处具有最大值，并且除了在310 m[mu]处的轻微凸起（其在发光反应中消失）之外与简单蛋白的紫外吸收类似，而在333 m[mu]处出现新的峰。总光随温度的升高而降低，酸度低于pH 5.1，碱度高于pH 8.3，而发射速率低于pH 6.5但高于pH 7.5时增加。在58[degree]的EDTA溶液中，或在83[degree]的饱和硫酸铵中，在1分钟内失去一半的活性。EDTA、丙二酸盐和脂肪族醛引起发光反应速率的可逆抑制，而芳香族醛、强氧化剂或还原剂和许多其他化学试剂仅降低总光。在水溶液中发光的最小组分是水母发光蛋白和Ca++。水母发光蛋白的分子量估计为35，000，并且在所涉及的条件下每个分子发射的总光为海萤属水母发光蛋白的一半。
Extraction with EDTA and purification by ammonium sulfate precipitation and column chromatography on DEAE yielded a substance, aequorin, with protein properties and high luminescence activity on addition of Ca++. Except for a questionable result with Sr++, no other cation was found to replace Ca++. No components active in light emission could be separated from aequorin by dialysis, ammonium sulfate precipitation, DEAE chromatography, or paper electrophoresis. The ultraviolet absorption of aequorin has a maximum at 280 m[mu] and is similar to that of simple proteins except for a slight bulge at 310 m[mu] which disappears in the luminescent reaction while a new peak appears at 333 m[mu]. Total light decreases with rise in temperature, with acidity below pH 5.1, and with alkalinity above pH 8.3, whereas the rate of emission decreases below pH 6.5 but increases above pH 7.5. Half of the activity is lost in 1 min at 58[degree] in EDTA solution, or at 83[degree]C in saturated ammonium sulfate. Reversible inhibition in rate of the luminescent reaction is caused by EDTA, malonate, and aliphatic aldehydes, whereas only the total light is decreased by aromatic aldehydes, strong oxidizing or reducing agents and a number of other chemical agents. The minimal components for luminescence in aqueous solution are aequorin and Ca++. The molecular weight of aequorin is estimated as 35,000, and total light emitted per molecule as half that of Cypridina luciferin under the conditions involved.