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

Absence o subs a es unde es ima es he measu emen o elec on anspo sys em ac i i y in zooplank on F. Maldonado-U ibe, T. Packa d and M. Gómez, Biological Oceanog aphy Labo a o y, Facul ad de Ciencias del Ma . Uni e sidad de Las Palmas de G an Cana ia, Campus Uni e si a io de Ta i a. 35017 Las Palmas de G.C., Cana y Islands, Spain. Respi a o y elec on anspo sys em (ETS) ac i i ies ha e been used o s udy oxygen consump ion in a my iad o ma ine o ganisms and communi ies all o e aqua ic en i onmen s. The me hodology a emp s o measu e he maximum eloci y o he ETS unde he hypo hesis is ha in ma ine plank on much o he physiology o espi a ion is con olled by enzymology (Packa d, 1971). Howe e , in he las decade an ETS a ia ion was de eloped elimina ing he biochemical eac an s (subs a es) ha makes he ETS assay speci ic o espi a o y oxygen consump ion. The p inciple behind his p ac ice was o measu e in i o ETS ac i i y and hence in-si u espi a ion (Båms ed , 2000). This assay used he e azolium sal , INT, o de ec elec on lux, bu did no use he subs a es (NADH, NADPH and succina e) equi ed o he ETS o unc ion. Ins ead i elied on he subs a es p esen in cy oplasm in he cell- ee ex ac o he sample. Un o una ely dilu ion du ing he ex ac p epa a ion educes he concen a ions o hese subs a es a below hei in i o le el and below he le el equi ed by he ETS enzymes. He e we examine his ze o-subs a e me hod by compa ing i o ETS ac i i y measu ed by s anda d me hods so as he capabili y o some compounds p esen in he cells o educe he INT in a non enzyma ic way. Tes s we e pe o med wi h wild adul males o L. ling u a. ETS ac i i y was measu ed by wo me hods ha use subs a es and he one me hod ha does no (Fig 1). The ze o- subs a e ETS me hod displayed weak ac i i y (Fig 1B), compa able o he s anda d me hod blanks. As a esul , his assay is much less sensi i e han he s anda d assay. Fu he mo e, i was no clea whe he e en his weak ac i i y was caused by he ETS o whe he i was caused by non-enzyma ic edox eac ions be ween INT and educing agen s in mysid cells. To es he possibili y, na u al educing agen s (asco bic acid, i amin-B12, glu a hione, cys eine, glucose and phenol), we e challenged o educe INT (Fig 2). Cys eine, glu a hione, and i amin-B12 all educed INT non enzyma ically as does asco bic acid (Fig 3). Thus hese subs ances in zooplank on samples will con use he in e p e a ion o ETS ac i i y measu emen s made wi hou subs a e addi ion. These subs ances a e ubiqui ous in ma ine o ganisms, gua an eeing con usion. 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 Abso bance (490nm) o 0,25 mM Abso bance (490 nm) Time (minu es) 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 o mazan·min-1by Vi C. µg o mazan·min-1 Concen a ion (mM) Glu a ion Cys ein Glucose Phenol Vi C Compound Slope ( a e) Glucose -0,00006 Phenol -0,00001 Glu a ion 0,0457 Cys eine 0,0641 Vi amin C 2,969 Lep omysis ling u a 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 p o ein (sample) ETS ac i i y on blank measu emen s e sus Båms ed essay Kenne & Ahmed BLANK Owens & King BLANK Bams ed 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 p o ein (sample) Kenne & Ahmed Owens & King Bams ed n= 13 n= 14 n= 57 Fig 1.: mic omola O2 pe hou espi a ion by 3 echniques, 2 o hem wi h sa u a ing subs a es (Kenne & Ahmed and Owens & King) whe e a la ge di e ence in he O2 consump ion signal is egis e ed. The signal o he ze o-subs a e echnique is equal o he blanks o he o he me hods (blowup (Fig.1B) Fig. 3. Righ side: Non enzyma ic o mazan p oduc ion by di e en concen a ions o asco bic acid. Le side: La ge scale o highis concen a ion o asco ba e (0,25 mM). The same ype o eac ion was ound o cys eine and glu a hione bu wi h a weake signal. Fig. 2. Fo mazan p oduc ion, non enzyma ically, by na u al compounds. Vi amine-C, cys eine, glu a hione, and i amin-B12 (da a no shown) educe INT. Glucose and phenol do no eac . Fo mazan p oduc ion o asco bic acid is ep esen ed on he axis o he le . Table inse shows he slopes. BÅMSTEDT, U. 2000. A New Me hod o Es ima e Respi a ion Ra e o Biological Ma e ial Based on he Reduc ion o Te azolium Viole . Jou nal o Expe imen al Ma ine Biology and Ecology. 251: 239-263. KENNER, R. & AHMED, S. 1975. Measu emen s o elec on anspo ac i i ies in ma ine phy oplank on. Ma ine Biology. 33: 119-127. OWENS, T. & KING, F. 1975. The Measu emen o Respi a o y Elec on T anspo Sys em Ac i i y in Ma ine Zooplank on. Ma ine Biology. 30: 27-36. PACKARD, T.T. 1971. The Measu emen o Respi a o y Elec on T anspo Ac i i y in Ma ine Phy oplank on. Jou nal o Ma ine Resea ch. 29: 235-244. F om ou esul s we conclude ha : 1,. The e is a di e ence o wo o de s o magni ude be ween he ETS a es o he me hods using subs a es [Owens & King (1975) and Kenne & Ahmed (1975)] and he one ha omi s hem (Båms ed , 2000). The ou pu o his las me hod was equal o he blanks o he i s wo me hods. 2.- The me hod sugges ed by Båms ed (2000) is no speci ic o he ETS. Why? Because INT can eac wi h many subs ances p esen in he cell ha may o may no be ela ed o espi a o y elec on anspo . Wha is measu ed is he backg ound educing capaci y o he ex ac ed cy oplasm. This educing capaci y is esponsible o he o mazan p oduc ion. 3.- Th ee o he six subs ances (glu a hione, cys eine and asco bic acid), eac ed s ongly and non-enzyma ically wi h INT, u he mo e, i amin B12 likely has he same po en ial o educe INT as asco bic acid ( i amin C).