Exploring ETS activity as a measure of potential respiration

Exploring ETS activity as a measure of potential respiration T.T. Packard*# , M. Alcaraz*, y M. Gómez *Instituto de Ciencias del Mar, Paseo Marítimo de la Barceloneta 37-49, 08003 Barcelona, Spain Biological Oceanography Laboratory, Facultad de Ciencias del Mar, Universidad de Las Palmas de Gran Canaria, Campus Universitario de Tafira., 35017 Las Palmas de G.C., Canary Islands, Spain #Bigelow Laboratory for Ocean Sciences, W. Boothbay Harbor, Maine 04538, USA. INTRODUCTION Is the ocean net autotrophic or heterotrophic? Is the biosphere losing nitrogen to anammox and denitrification or is it in balance with nitrogen fixation and anthropogenic inputs? These questions cannot be resolved without accurate measurements of the physiological processes involved and yet our methodology lags far behind our curiosity and the demand to monitor our rapidly changing planet. This situation cries out for methods development. For this reason, as in Gomez et al (1996), we continue to explore the use of the respiratory electron transport system (ETS) as a measure of potential respiration ( ) and a proxy for respiration (R). Here we demonstrate: (1) how kinetic ETS assays document reaction-rate time-courses, cuts reaction-time in half, and eliminates turbidity blanks; (2) why substrate blanks do not measure ETS activity and why a Michaelis-Menten Vmax should be measured instead; and (3) the biochemical meaning of R/ in seawater. We also present new data on: (4) the molar specific absorption coefficient for the tetrazoliums, INT and CTC (17.8 and 22.9 absorbance units (mM INT formazan)-1 (1 cm cuvette) -1, respectively). Finally we propose using baker’s yeast (Saccharomyces cerviseae), with an ETS activity (20°C) of 25.1 ± 5.1 mol emin-1 (mg dry weight)-1 as a biological standard. Endpoint Analysis Pitfalls 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 Time (minutes) Potential Respiration (nM O2 min-1 sampleAccelerating Rate Decelerating Rate Instantaneous Reaction Delayed Instantaneous Reaction Reactant Unlimited Linear Reactant Limited Reaction Conventional Analysis considered only the initial and the final points. There was no way to detect an error in the reaction. Fig. 1A TURBIDITY BLANK needed because the measurements were made on different solutions with potentially varying background turbidity. INITIAL VALUES FINAL VALUES Two separate Solutions needed! 20 min Time Course reveals errors! 0 10 20 30 40 50 0 5 10 15 20 25 Time (minutes) Pot. Resp. (nM O2 min-1 sampleAccelerating Rate Decelerating Rate Instantaneous Reaction Delayed Instantaneous Reaction Reactant Unlimited Linear Reactant Limited Reaction Actual reaction behavour invisable to "Endpoint" analysis. Only the "Reactant Unlimited Linear" curve is valid. The others are errors! Fig. 1B TURBIDITY BLANK (Not needed now.) Only one solution needed! 8 min Time Course saves time & reveals errors! 0 5 10 15 20 25 0 2 4 6 8 10 Time (minutes) Potential Respiration (nM O2 min-1 sampleAccelerating Rate Decelerating Rate Instantaneous Reaction Delayed Instantaneous Reaction Reactant Unlimited Linear Reactant Limited Reaction Objective: "Reactant Unlimited Linear" curve. Others contain errors. Fig. 1C TURBIDITY BLANK not needed because the same solution is used for all measurements and rate is the slope of the line! Only one solution needed! Gyrodinium corsicum ETS y = 0,0765x + 0,2602 R2 = 0,9992 y = 0,0007x + 0,2207 R2 = 0,9107 0,2 0,4 0,6 0,8 0 1 2 3 4 5 6 Time (minutes) Absorbance (450nm) 080599 ICM Barcelona Substrate added No substrate added Fig. 2A METHODS ETS assays reported here are based on the Packard and Williams (1981) method with the simplifications: (1) that turbidity and substrate blanks have been replaced by a reagent blank (an ETS assay with <1/1000 part of the sample’s biological material); (2) that succinate as a reactant (Savenkoff et al, 1995) and cyanide as an inhibitor of cytochrome oxidase, have been eliminated. All measurements were made kinetically according to F. D. King (personal communication) with a computer-controlled spectrophotometer using automatic sample changing and temperature control. Homogenates were kept at 0-4°C and were measured within 20 min after being removed from either liquid nitrogen or -80° storage. RESULTS Fig.1A-1C show that kinetic analysis offers the advantages of: (1) detecting errors during the ETS reaction; (2) halving the time of the analysis; (3) increased sensitivity and resolution through better slope definition and higher data acquisition rate. Figs. 2A-2C shows that ETS activity cannot be measured in zooplankton, phytoplankton, or seawater (microplankton) without adding substrates (NADH and NADPH). Fig. 3 illustrates why an irreproducible result is obtained if substrate is not added to an enzyme assay based on disrupted cells. Fig. 4 compares the currently used reagent blank with an ETS assay done on a seawater sample. To reconcile known variability in the ratio of respiration (R) to potential respiration ( ) in bacteria (Fig. 5A and 5B) with the use of a constant R/ in oceanographic fieldwork (Fig. 5C) one can consider a kinetic formulation of respiration (Box 1). Upon doing this, it becomes apparent that R/ in the field (Arístegui and Montero, 1995) can be an oceanographic average of the expression, [NADPH][NADH]/(Kb+[NADPH][NADH]), as explained in Box 1. This is an unproven, but testable hypothesis. ETS activity, as currently measured, is based on a specific absorbance (A490) of 15.9 absorbance units mM-1 in a 1 cm cuvette of the INT formazan at a wavelength of 490nm. Here we show that the peak at 490 nm in phosphate buffer is correct (Fig. 6A), but that A490 may be 17.8 (Fig. 6B) rather than 15.9. We also tested another tetrazolium salt (CTC) to see if it could replace INT as an electron acceptor. We found its absorption maximum at 455nm (Fig. 6C) and its A455 to be 22.9 absorbance units mM-1 (Fig. 6D). CONCLUSIONS and SUMMARY 1. Kinetic analysis provides more sensitivity, reliability, information, and a higher data acquisition rate than end point analysis (Figs. 1A-1C). 2. An ETS assay measured without adding substrates (substrate blank) yields practically no activity and therefore cannot serve as a measure of . Furthermore, it is not a measure of invivo ETS activity because the reactants, during homogenization, are driven far lower than their occurrence in the living organism (Figs. 2-4). 3. Fig. 5 and Box 1 argue that a measurement of R/ in the ocean is a measure of [NADPH][NADH]/(Kb+[NADPH][NADH]) in the plankton. 4. Fig. 6 supports the use of INT as an efficient electron acceptor for the ETS. 5. Fig. 7 illustrates the facility of using yeast as a biological standard for ETS measurements. Microplankton ETS Activity y = 0,0081x + 0,1997 R2 = 0,9997 y = -1E-05x + 0,181 R2 = 0,0032 0,15 0,20 0,25 0 1 2 3 4 5 6 Time (minutes) Absorbance (490 nm) 080599 Barcelona Harbor (seawater GF/F filtered) Substrate added No substrate added Fig. 2C ETS Activity Zooplankton y = 0,0202x + 0,139 R2 = 0,9999 y = 0,0003x + 0,1137 R2 = 0,8491 0,00 0,05 0,10 0,15 0,20 0,25 0,30 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 Time (minutes) Absorbance (490 nm) No substrate added! Substrates added! Sample 57A Fig. 2B Reagent Blank: Its Better (Seawater example) 0,1 0,2 0,3 0,4 0,5 0 1 2 3 4 5 6 7 8 Time (minutes) Absorbance (490 nm, 1 cm cuvette) Average slope = 0.0038 OD units/min Average slope = 0.0277 OD units/min Blanes Time Series Station 16 July 2003 Fig. 4 ETS Assay Reagent Blank R/ ETS by Arístegui & Montero (1995) y = 0,5949x + 16,223 R2 = 0,8967 0 40 80 120 160 200 050 100 150 200 250 ETS Activity (mg O2 m-3 dayRespiration (mg O2 m-3 day7 expeditions to different parts of the world! R/F = 0.59 ± 0.20 Fig. 5C R/ETS time course 0,00 0,10 0,20 0,30 0,40 0 5 10 15 20 25 30 TIME (hours) RATIO of Resp to ETS Activity Pseudomonas nautica, batch culture on pyruvate NUTRIENT DEPLETION Fig. 5B ETS & Respiration time course (Packard et al., 1995) 0 50 100 150 200 250 300 0 5 10 15 20 25 30 TIME (hours) ETS & Respiration Pseudomonas nautica, batch culture on pyruvate ETS ACTIVITY OXYGEN CONSUMPTION NUTRIENT DEPLETION Fig. 5A Determination of INT's absorptivity y = 0,0178x + 0,094 R2 = 0,9953 0,0 0,2 0,4 0,6 0,8 0 5 10 15 20 25 30 35 40 Ascorbate Concentration (micromolar) Absorbance (490 nm, 1 cm cuvette) 281299 Fig. 6B Absorption Spectrum 0,8 0,9 1,0 1,1 1,2 420 440 460 480 500 520 540 Wavelength (nm) Absorbance (1 cm cuvette) Normal Measurement at 490 nm Fig 6A Determination of CTC's absorptivity y = 0,0229x + 0,0733 R2 = 0,9966 0,0 0,2 0,4 0,6 0 5 10 15 20 Ascorbate Concentration (micromolar) Absorbance (455 nm) 281299 Fig. 6D Absorbance CTC's Formazan 0,6 0,8 1,0 420 440 460 480 500 Wavelength (nm) Absorbance (1 cm cuvette) Normal measuremen t at 455nm Fig. 6D Yeast ETS Activity (Saccharomyces cerviseae) 0 0,1 0,2 0,3 0,4 0,5 4 5 6 7 8 Time (minutes) Absorbance (490nm, 1 cm cuvette) yb260903 Reagent Blanks. Ave = 0.004 Assays in triplicate. Ave = 0.056 ± 0.004 Fig. 7 If respiration (R) in seawater, phytoplankton, bacterioplankton, and zooplankton can be represented by the equation: R = [NADPH][NADH]/(Kb+[NADPH][NADH]), where = potential respiration, NADH and NADPH are the principal reactants (substrates) for the ETS, and Kb = (KNADH)(Kia) + (KNADPH)[NADH] + (KNADH)[NADPH], where Kia is the dissociation constant for the ETS-NADPH complex, KNADH is the affinity of the ETS for NADH, and KNADPH is the affinity of the ETS for NADPH; Then for seawater, reconciliation between Figs. 5A, 5B, and 5C is found when [NADPH][NADH]/(Kb+[NADPH][NADH]) for seawater equals the slope of the calibration plot between seawater R and seawater ! In other words for a seawater average (Fig. 5C) according to Arístegui & Montero (1995), [NADPH][NADH]/(Kb+[NADPH][NADH]) = 0.59 Box 1 Box 1 REFERENCES Arístegui, J.; Montero, M.F. 1995. 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