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).