Absence of substrates underestimates the measurement of electron transport system activity in zooplankton

Absence of substrates underestimates the measurement of electron transport system activity in zooplankton F. Maldonado-Uribe, T. Packard and M. Gómez, 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. Respiratory electron transport system (ETS) activities have been used to study oxygen consumption in a myriad of marine organisms and communities all over aquatic environments. The methodology attempts to measure the maximum velocity of the ETS under the hypothesis is that in marine plankton much of the physiology of respiration is controlled by enzymology (Packard, 1971). However, in the last decade an ETS variation was developed eliminating the biochemical reactants (substrates) that makes the ETS assay specific for respiratory oxygen consumption. The principle behind this practice was to measure invivo ETS activity and hence in-situ respiration (Båmstedt, 2000). This assay used the tetrazolium salt, INT, to detect electron flux, but did not use the substrates (NADH, NADPH and succinate) required for the ETS to function. Instead it relied on the substrates present in cytoplasm in the cell-free extract of the sample. Unfortunately dilution during the extract preparation reduces the concentrations of these substrates far below their invivo level and below the level required by the ETS enzymes. Here we examine this zero-substrate method by comparing it to ETS activity measured by standard methods so as the capability of some compounds present in the cells to reduce the INT in a non enzymatic way. Tests were performed with wild adult males of L. lingvura. ETS activity was measured by two methods that use substrates and the one method that does not (Fig 1). The zerosubstrate ETS method displayed weak activity (Fig 1B), comparable to the standard method blanks. As a result, this assay is much less sensitive than the standard assay. Furthermore, it was not clear whether even this weak activity was caused by the ETS or whether it was caused by non-enzymatic redox reactions between INT and reducing agents in mysid cells. To test the possibility, natural reducing agents (ascorbic acid, vitamin-B12, glutathione, cysteine, glucose and phenol), were challenged to reduce INT (Fig 2). Cysteine, glutathione, and vitamin-B12 all reduced INT non enzymatically as does ascorbic acid (Fig 3). Thus these substances in zooplankton samples will confuse the interpretation of ETS activity measurements made without substrate addition. These substances are ubiquitous in marine organisms, guaranteeing confusion. 0 0,5 1 1,5 2 2,5 3 0 0,2 0,4 0,6 0,8 1 1,2 1,4 0 1 2 3 4 5 Absorbance (490nm) for 0,25 mM Absorbance (490 nm) Time (minutes) ASCORBIC ACID 1E-4 mM 5E-4 mM 1E-3 mM 5E-3 mM 0,01 mM 0,05 mM 0,25 mM 0 1 2 3 4 5 6 7 0 0,04 0,08 0,12 0,16 0,2 0 0,25 0,5 0,75 1 1,25 1,5 1,75 µg formazan·min-1by Vit C. µg formazan·min-1 Concentration (mM) Glutation Cystein Glucose Phenol Vit C Compound Slope (rate) Glucose -0,00006 Phenol -0,00001 Glutation 0,0457 Cysteine 0,0641 Vitamin C 2,969 Leptomysis lingvura y = 4E-05x - 0,0025 R² = 0,5324 y = 3E-05x + 0,0031 R² = 0,4807 y = 1E-05x + 0,011 R² = 0,0481 0,000 0,005 0,010 0,015 0,020 0,025 0,030 0 100 200 300 400 500 600 700 800 µmol O2 · h-1 µg protein (sample) ETS activity on blank measurements versus Båmstedt essay Kenner & Ahmed BLANK Owens & King BLANK Bamstedt y = 0,0006x - 0,0226 R² = 0,6159 y = 0,0011x + 0,0073 R² = 0,7461 y = 1E-05x + 0,011 R² = 0,0481 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0 100 200 300 400 500 600 700 800 µmol O2 · h-1 µg protein (sample) Kenner & Ahmed Owens & King Bamstedt n= 13 n= 14 n= 57 Fig 1.: micromolar O2 per hour respiration by 3 techniques, 2 of them with saturating substrates (Kenner & Ahmed and Owens & King) where a large difference in the O2 consumption signal is registered. The signal of the zero-substrate technique is equal to the blanks of the other methods (blowup (Fig.1B) Fig. 3. Right side: Non enzymatic formazan production by different concentrations of ascorbic acid. Left side: Larger scale for highist concentration of ascorbate (0,25 mM). The same type of reaction was found for cysteine and glutathione but with a weaker signal. Fig. 2. Formazan production, non enzymatically, by natural compounds. Vitamine-C, cysteine, glutathione, and vitamin-B12 (data not shown) reduce INT. Glucose and phenol do not react. Formazan production of ascorbic acid is represented on the axis to the left. Table inset shows the slopes. BÅMSTEDT, U. 2000. A New Method to Estimate Respiration Rate of Biological Material Based on the Reduction of Tetrazolium Violet. Journal of Experimental Marine Biology and Ecology. 251: 239-263. KENNER, R. & AHMED, S. 1975. Measurements of electron transport activities in marine phytoplankton. Marine Biology. 33: 119-127. OWENS, T. & KING, F. 1975. The Measurement of Respiratory Electron Transport System Activity in Marine Zooplankton. Marine Biology. 30: 27-36. PACKARD, T.T. 1971. The Measurement of Respiratory Electron Transport Activity in Marine Phytoplankton. Journal of Marine Research. 29: 235-244. From our results we conclude that: 1,. There is a difference of two orders of magnitude between the ETS rates of the methods using substrates [Owens & King (1975) and Kenner & Ahmed (1975)] and the one that omits them (Båmstedt, 2000). The output of this last method was equal to the blanks of the first two methods. 2.- The method suggested by Båmstedt (2000) is not specific for the ETS. Why? Because INT can react with many substances present in the cell that may or may not be related to respiratory electron transport. What is measured is the background reducing capacity of the extracted cytoplasm. This reducing capacity is responsible for the formazan production. 3.- Three of the six substances (glutathione, cysteine and ascorbic acid), reacted strongly and non-enzymatically with INT, furthermore, vitamin B12 likely has the same potential to reduce INT as ascorbic acid (vitamin C).