Measuring Bioluminescence in the Field

Coelenterazine and the corresponding luciferase can be easily tested in the field. A small piece of tissue sample is put in a test tube with methanol (for coelenterazine) or water (for luciferase), and crushed with a spatula. To measure coelenterazine, a buffer solution containing a coelenterazine luciferase is injected into a small amount of the fluid part of the crushed sample mixture. Similarly, luciferase can be measured with a buffer solution containing coelenterazine. The presence of Cypridina luciferin can be tested in the same fashion, with the methanol extract of samples and crude Cypridina luciferase. However, the detection of a very weak Cypridina luciferase activity in the field is not recommended (see Section C5.6). To test the presence of a Ca2+-sensitive photoprotein, crush a sample in a neutral buffer solution containing 20-50 mM EDTA, and then add lOmM calcium acetate to a small portion of the fluid part of the crushed sample to detect any light emission. 

Considering the effects of the four factors described above, the amount of luciferin required to obtain highly reliable data would be 

The system is continuously evacuated to a pressure of about 1 pm Hg (0.133Pa) without coolant of the traps for at least 2hr before 

At this point, two options are available if required. (1) If more complete degassing is desired, freeze the liquid contents by soaking the appropriate part of reaction vessel in dry ice/acetone bath, evacuate to 10 pm Hg, then thaw the contents, followed by an additional 10 min degassing. (2) If the removal of residual 1602 is desired, mix the two solutions by tilting the vessel to consume the 1602 by luciferin-luciferase luminescence reaction. The resulting reaction will cease in 2-3 min. Then, degas the content for 1-2 min to remove the C02 produced in the luminescence reaction. 

Calculations. The atoms of incorporated lsO is calculated from mass spectral data. When the ratio of the peak heights at mass-to-charge ratio (m/e) 44, 46 and 48 for CO2 is X-.Y-.Z, assuming that the height of each peak is strictly proportional to the number of CO2 molecules, the atom fraction of l80 in CO2, C, is given by  

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