Application of adenylate energy charge (EC) measurements in marine ecological studies requires high accuracy and precision. We have examined the luciferinluciferase method for EC determination and suggest the following methodological improvements. Replacement of the commonly used potassium phosphate buffer with potassium chloride in the assay medium prevents problems due to precipitation of magnesium phosphates. Interference from ADP and AMP in crude firefly extract can be eliminated by their enzymatic decomposition with apyrase and AMP-deaminase. Impure nucleotide standards are perhaps more common than currently realized, and can lead to inaccurate estimates of EC. proposed as an index of the energetical state of natural microbial communities and applied to both water and sediment samples (e. g. Wiebe and Bancroft, 1975;Witzel, 1979;Christensen and Devol, 1980;Karl, 1980;Romano and Daumas, 1981). EC measurements have also been used in studies of various types of stress in marine invertebrates (Ivanovici, 1980; Haya and Waiwood, in press). The EC ratio is normally stabilized at about 0.9 and changes in EC associated with environmental and physiological changes are usually small (Atkinson, 1977;Karl, 1980). High accuracy and precision in measurements are therefore important requirements for future evaluations of the usefulness of the EC ratio for ecological applications. We have examined the luciferin-luciferase methodology for EC determination with regard to potential interferences and standardization requirements.
The adenylate energy charge ratio (EC =[Fifty mg freeze-dried firefly lantern extract (FLE-50; Sigma) were rehydrated with 10 to 25 m1 distilled water and aged successively for 4 and 12 to 48 h at room temperature and refrigerated, respectively. The preparation was cleared by centrifugation and warmed to room temperature prior to use. Enzymatic treatment of this FLE was performed by incubating a 5 rnl subsample with additions of 250 p1 AMP-deaminase (Sigma, Grade IV) and 0.5 mg apyrase (ATP-and ADPase; Sigma, Grade I) for 4 h at room temperature. Adenine nucleotides (AN) in the FLE preparations were extracted by injecting 0.5 m1 FLE into 4.5 m1 boiling Tris-buffer/EDTA (Lundin and Thore, 1975). ATP was assayed as described by Skjoldal and Barkati (1982). AMP and/or ADP were phosphorylated to ATP prior to assay by incubating (30 "C, 30 min) sample portions with phospho(eno1)pyruvate (PEP), adenylate kinase (AK) and/or pyruvate kinase (PK) (madet, 1967). Two different sets of incubation reagents, based on either potassium phosphate (KP,) buffer or potassium chloride (KCl), were compared in one experiment. In all other experiments the KC1-reagents were used. The KP, incubation method followed with slight modifications that of Chapman et al. (1971). To 250 p1 subsamples were added 50 p1 MgC1, (25 mM) and 50 p1 reagent. The reagent for determination of ATP (ATP-reagent) was KP,-buffer (K2HP0,/KH2P0,, 75 mM, pH 7.5). The reagent for determination of ATP + ADP (ADP-reagent) contained KP,-buffer,...