Purification and Molecular Properties of Cypridina Luciferase

Purification of luciferase. Cypridina luciferase is more stable than many other luciferases, except that this enzyme is rapidly inactivated at acidity below pH 5.0. The dried specimens that have been stored for over 50 years at room temperature (sometimes exceeding 30°C) still possess strong luciferase activity that can be extracted and purified. Preparations of highly purified luciferase have been obtained by various methods (McElroy and Chase, 1951 Shimomura et al., 1961,1969 Tsuji and Sowinski, 1961 Stone, 1968 Tsuji etal., 1974 Thompson et al., 1989) the purification methods employed include 

Molecular weight. The molecular weight of C. hilgendorfii luciferase reported in the past varies considerably across a range of 50,000-80,000 (Chase and Langridge, 1960 Shimomura etal., 1961, 1969 Tsuji and Sowinski, 1961 Tsuji et al., 1974) it appears most likely to be 60,000-70,000. The luciferase is an acidic protein with an isoelectric point of 4.35 (Shimomura et al., 1961). The absorption spectrum of luciferase is that of a simple protein without any prosthetic group, showing a peak at 280 nm. Absorbance value at 280 nm of a 0.1% luciferase solution is approximately 0.96 (Shimomura etal., 1969). 

Inhibitors. Many common enzyme inhibitors show little or no effect on the activity of Cypridina luciferase in the luminescence reaction (Tsuji et al., 1974). However, EDTA strongly inhibits the bioluminescence reaction, showing a peculiar relationship between the 

EDTA concentration and the degree of inhibition (Shimomura et al., 1961). In a 30 mM sodium phosphate buffer containing 60 mM NaCl, pH 7.0, inhibition by EDTA is very strong (84%) even at an EDTA concentration as low as 25 pM, but the inhibition does not increase beyond 90% even at an EDTA concentration of 5 mM. The inhibition is completely reversed by the addition of a sufficient amount of Ca2+ to bind EDTA. 

Why does EDTA cause only 90% inhibition, leaving 10% of the activity intact Buffer solutions usually contain 0.1 1 pM of contaminating Ca2+ when special precaution is not taken, and this concentration is much greater than the molar concentration of luciferase used in the experiments. Thus, one of the possibilities would be that Ca2+ interacts with the molecule of luciferase and can increase the activity of luciferase about 10 times, in spite of the fact that the molecule of luciferase lacks the Ca2+ binding site of EF-hand type (Thompson et al., 1989). Another possibility would be that EDTA interacts directly with the molecules of luciferase, to cause the inhibition. The question remains unresolved. 

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