. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 0 10 20 30 40 50 60 Temperature (°C) Figure 4. Effect of temperature on the luminescence intensities of coelenterazine catalyzed by luciferases A. B. C. and L. The measurements were done in 20 mM Tris-HCl buffer (pH ), containing 1 M NaCl and BSA (the standard buffer)- The luminescence reaction was started by the addition of 10 /al of mM methanolic coelenterazine. The amount of sample used for measuring each point: luciferase A, 170 LU; luciferase B, 190 LU: luciferase C, 210 LU; luciferase L, 210 LU. Luminescen
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 0 10 20 30 40 50 60 Temperature (°C) Figure 4. Effect of temperature on the luminescence intensities of coelenterazine catalyzed by luciferases A. B. C. and L. The measurements were done in 20 mM Tris-HCl buffer (pH ), containing 1 M NaCl and BSA (the standard buffer)- The luminescence reaction was started by the addition of 10 /al of mM methanolic coelenterazine. The amount of sample used for measuring each point: luciferase A, 170 LU; luciferase B, 190 LU: luciferase C, 210 LU; luciferase L, 210 LU. Luminescence reaction of coelenterazine and its analogs catalyzed by luciferases A, B, and C The spectra of the luminescence of coelenterazine cata- lyzed by luciferase A, B, and C were all identical with that of luciferase L, showing a peak at 465 nm. The specific activity (quanta emitted per second, divided by A2SO nm. \ cm) of the materials obtained in Step 5, Table 1, was X 1016 photons/s for luciferase A, X 1016 photons/s for luciferase B, and X 1016 photons/s for luciferase C, under the standard assay conditions. However, significantly higher specific activities were obtained when the purifica- tion included the alternative method for Steps 1 and 2: X 1016 photons/s and X 10lh photons/s for luciferases A and B, respectively (the yield of luciferase C was low). The maximum specific activities obtainable with high concen- trations of coelenterazine (over 2 \iM) should be roughly twice these values, based on the data of Figure 8 (note that the coelenterazine concentration in the standard assay is about /u,Af). As a reference to these data, the maximum specific activity of luciferase L reported previously was 8 X 10'3 photons/s (Shimomura and Flood, 1998). The quantum yields of coelenterazine in the luminescence reaction cata- lyzed at 24 °C by luciferases A, B, and C were , , and , respectively, compared with previously reported for luc
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