Explo ing ETS ac i i y as a measu e o po en ial espi a ion
T.T. Packa d*# , M. Alca az*, y M. Gómez
*Ins i u o de Ciencias del Ma , Paseo Ma í imo de la Ba celone a 37-49, 08003 Ba celona, Spain 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 #Bigelow Labo a o y o Ocean Sciences, W. Boo hbay Ha bo ,
Maine 04538, USA.
INTRODUCTION Is he ocean ne au o ophic o he e o ophic? Is he biosphe e losing
ni ogen o anammox and deni i ica ion o is i in balance wi h ni ogen ixa ion and
an h opogenic inpu s? These ques ions canno be esol ed wi hou accu a e
measu emen s o he physiological p ocesses in ol ed and ye ou me hodology lags a
behind ou cu iosi y and he demand o moni o ou apidly changing plane . This
si ua ion c ies ou o me hods de elopmen . Fo his eason, as in Gomez e al (1996),
we con inue o explo e he use o he espi a o y elec on anspo sys em (ETS) as a
measu e o po en ial espi a ion ( ) and a p oxy o espi a ion (R).
He e we demons a e: (1) how kine ic ETS assays documen eac ion- a e ime-cou ses,
cu s eac ion- ime in hal , and elimina es u bidi y blanks; (2) why subs a e blanks do
no measu e ETS ac i i y and why a Michaelis-Men en Vmax should be measu ed ins ead;
and (3) he biochemical meaning o R/ in seawa e . We also p esen new da a on: (4)
he mola speci ic abso p ion coe icien o he e azoliums, INT and CTC (17.8 and
22.9 abso bance uni s (mM INT o mazan)-1 (1 cm cu e e) -1, espec i ely). Finally we
p opose using bake ’s yeas (Saccha omyces ce iseae), wi h an ETS ac i i y (20°C) o
25.1 ± 5.1 mol e- min-1 (mg d y weigh )-1 as a biological s anda d.
Endpoin Analysis Pi alls
0
5
10
15
20
25
30
35
40
45
50
0 5 10 15 20 25
Time (minu es)
Po en ial Respi a ion
(nM O2 min-1 sample-
Accele a ing Ra e
Decele a ing Ra e
Ins an aneous Reac ion
Delayed Ins an aneous Reac ion
Reac an Unlimi ed Linea
Reac an Limi ed Reac ion
Con en ional Analysis conside ed
only he ini ial and he inal poin s.
The e was no way o de ec an e o
in he eac ion.
Fig. 1A
TURBIDITY BLANK needed because he measu emen s we e made
on di e en solu ions wi h po en ially a ying backg ound u bidi y.
INITIAL VALUES
FINAL
VALUES
Two sepa a e
Solu ions needed!
20 min Time Cou se e eals e o s!
0
10
20
30
40
50
0 5 10 15 20 25
Time (minu es)
Po . Resp. (nM O2 min-1 sample-
Accele a ing Ra e
Decele a ing Ra e
Ins an aneous Reac ion
Delayed Ins an aneous Reac ion
Reac an Unlimi ed Linea
Reac an Limi ed Reac ion
Ac ual eac ion beha ou
in isable o "Endpoin " analysis.
Only he "Reac an Unlimi ed
Linea " cu e is alid. The
o he s a e e o s!
Fig. 1B
TURBIDITY BLANK (No needed now.)
Only one solu ion
needed!
8 min Time Cou se sa es ime & e eals e o s!
0
5
10
15
20
25
0 2 4 6 8 10
Time (minu es)
Po en ial Respi a ion
(nM O2 min-1 sample-
Accele a ing Ra e
Decele a ing Ra e
Ins an aneous Reac ion
Delayed Ins an aneous Reac ion
Reac an Unlimi ed Linea
Reac an Limi ed Reac ion
Objec i e: "Reac an Unlimi ed Linea "
cu e. O he s con ain e o s.
Fig. 1C
TURBIDITY BLANK no needed because he same solu ion is used
o all measu emen s and a e is he slope o he line!
Only one solu ion
needed!
Gy odinium co sicum 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 (minu es)
Abso bance (450nm)
080599 ICM Ba celona
Subs a e added
No subs a e added
Fig. 2A
METHODS ETS assays epo ed he e a e based on he Packa d and Williams (1981)
me hod wi h he simpli ica ions: (1) ha u bidi y and subs a e blanks ha e been
eplaced by a eagen blank (an ETS assay wi h <1/1000 pa o he sample’s biological
ma e ial); (2) ha succina e as a eac an (Sa enko e al, 1995) and cyanide as an
inhibi o o cy och ome oxidase, ha e been elimina ed. All measu emen s we e made
kine ically acco ding o F. D. King (pe sonal communica ion) wi h a compu e -con olled
spec opho ome e using au oma ic sample changing and empe a u e con ol.
Homogena es we e kep a 0-4°C and we e measu ed wi hin 20 min a e being emo ed
om ei he liquid ni ogen o -80° s o age.
RESULTS Fig.1A-1C show ha kine ic analysis o e s he ad an ages o : (1) de ec ing
e o s du ing he ETS eac ion; (2) hal ing he ime o he analysis; (3) inc eased
sensi i i y and esolu ion h ough be e slope de ini ion and highe da a acquisi ion a e.
Figs. 2A-2C shows ha ETS ac i i y canno be measu ed in zooplank on, phy oplank on,
o seawa e (mic oplank on) wi hou adding subs a es (NADH and NADPH). Fig. 3
illus a es why an i ep oducible esul is ob ained i subs a e is no added o an enzyme
assay based on dis up ed cells. Fig. 4 compa es he cu en ly used eagen blank wi h an
ETS assay done on a seawa e sample.
To econcile known a iabili y in he a io o espi a ion (R) o po en ial espi a ion ( )
in bac e ia (Fig. 5A and 5B) wi h he use o a cons an R/ in oceanog aphic ieldwo k
(Fig. 5C) one can conside a kine ic o mula ion o espi a ion (Box 1). Upon doing his,
i becomes appa en ha R/ in he ield (A ís egui and Mon e o, 1995) can be an
oceanog aphic a e age o he exp ession, [NADPH][NADH]/(Kb+[NADPH][NADH]),
as explained in Box 1. This is an unp o en, bu es able hypo hesis.
ETS ac i i y, as cu en ly measu ed, is based on a speci ic abso bance (A490) o 15.9
abso bance uni s mM-1 in a 1 cm cu e e o he INT o mazan a a wa eleng h o 490nm.
He e we show ha he peak a 490 nm in phospha e bu e is co ec (Fig. 6A), bu ha
A490 may be 17.8 (Fig. 6B) a he han 15.9. We also es ed ano he e azolium sal
(CTC) o see i i could eplace INT as an elec on accep o . We ound i s abso p ion
maximum a 455nm (Fig. 6C) and i s A455 o be 22.9 abso bance uni s mM-1 (Fig. 6D).
CONCLUSIONS and SUMMARY
1. Kine ic analysis p o ides mo e sensi i i y, eliabili y, in o ma ion, and a highe
da a acquisi ion a e han end poin analysis (Figs. 1A-1C).
2. An ETS assay measu ed wi hou adding subs a es (subs a e blank) yields
p ac ically no ac i i y and he e o e canno se e as a measu e o .
Fu he mo e, i is no a measu e o in i o ETS ac i i y because he eac an s,
du ing homogeniza ion, a e d i en a lowe han hei occu ence in he li ing
o ganism (Figs. 2-4).
3. Fig. 5 and Box 1 a gue ha a measu emen o R/ in he ocean is a measu e o
[NADPH][NADH]/(Kb+[NADPH][NADH]) in he plank on.
4. Fig. 6 suppo s he use o INT as an e icien elec on accep o o he ETS.
5. Fig. 7 illus a es he acili y o using yeas as a biological s anda d o ETS
measu emen s.
Mic oplank on ETS Ac i i y
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 (minu es)
Abso bance (490 nm)
080599 Ba celona Ha bo
(seawa e GF/F il e ed)
Subs a e added
No subs a e added
Fig. 2C
ETS Ac i i y
Zooplank on
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 (minu es)
Abso bance (490 nm)
No subs a e added!
Subs a es added!
Sample 57A
Fig. 2B
Reagen Blank: I s Be e (Seawa e example)
0,1
0,2
0,3
0,4
0,5
0 1 2 3 4 5 6 7 8
Time (minu es)
Abso bance (490 nm,
1 cm cu e e)
A e age slope = 0.0038 OD uni s/min
A e age slope = 0.0277 OD uni s/min
Blanes Time
Se ies S a ion
16 July 2003
Fig. 4
ETS Assay
Reagen Blank
R/ ETS by A ís egui & Mon e o (1995)
y = 0,5949x + 16,223
R2 = 0,8967
0
40
80
120
160
200
050 100 150 200 250
ETS Ac i i y (mg O2 m-3 day-
Respi a ion (mg O2 m-3 day-
7 expedi ions o di e en pa s o he wo ld!
R/F = 0.59 ± 0.20
Fig. 5C
R/ETS ime cou se
0,00
0,10
0,20
0,30
0,40
0 5 10 15 20 25 30
TIME (hou s)
RATIO o Resp o ETS
Ac i i y
Pseudomonas
nau ica, ba ch
cul u e on py u a e
NUTRIENT
DEPLETION
Fig. 5B
ETS & Respi a ion ime cou se
(Packa d e al., 1995)
0
50
100
150
200
250
300
0 5 10 15 20 25 30
TIME (hou s)
ETS & Respi a ion
Pseudomonas
nau ica, ba ch
cul u e on
py u a e
ETS
ACTIVITY
OXYGEN
CONSUMPTION
NUTRIENT
DEPLETION
Fig. 5A
De e mina ion o INT's abso p i i y
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
Asco ba e Concen a ion (mic omola )
Abso bance (490
nm, 1 cm cu e e)
281299
Fig. 6B
Abso p ion
Spec um
0,8
0,9
1,0
1,1
1,2
420 440 460 480 500 520 540
Wa eleng h (nm)
Abso bance (1 cm cu e e)
No mal
Measu emen
a 490 nm
Fig 6A
De e mina ion o CTC's abso p i i y
y = 0,0229x + 0,0733
R2 = 0,9966
0,0
0,2
0,4
0,6
0 5 10 15 20
Asco ba e Concen a ion (mic omola )
Abso bance (455 nm)
281299
Fig. 6D
Abso bance
CTC's Fo mazan
0,6
0,8
1,0
420 440 460 480 500
Wa eleng h (nm)
Abso bance (1 cm cu e e)
No mal
measu emen
a 455nm
Fig. 6D
Yeas ETS Ac i i y
(Saccha omyces ce iseae)
0
0,1
0,2
0,3
0,4
0,5
4 5 6 7 8
Time (minu es)
Abso bance (490nm, 1
cm cu e e)
yb260903
Reagen Blanks. A e = 0.004
Assays in
iplica e. A e =
0.056 ± 0.004
Fig. 7
I espi a ion (R) in seawa e , phy oplank on, bac e ioplank on, and
zooplank on can be ep esen ed by he equa ion:
R = [NADPH][NADH]/(Kb+[NADPH][NADH]),
whe e = po en ial espi a ion, NADH and NADPH a e he p incipal eac an s
(subs a es) o he ETS, and
Kb = (KNADH)(Kia) + (KNADPH)[NADH] + (KNADH)[NADPH],
whe e Kia is he dissocia ion cons an o he ETS-NADPH complex, KNADH is he
a ini y o he ETS o NADH, and KNADPH is he a ini y o he ETS o NADPH;
Then o seawa e , econcilia ion be ween Figs. 5A, 5B, and 5C is ound when
[NADPH][NADH]/(Kb+[NADPH][NADH]) o seawa e equals he slope o he
calib a ion plo be ween seawa e R and seawa e ! In o he wo ds o a seawa e
a e age (Fig. 5C) acco ding o A ís egui & Mon e o (1995),
[NADPH][NADH]/(Kb+[NADPH][NADH]) = 0.59 Box 1
Box 1
REFERENCES
A ís egui, J.; Mon e o, M.F. 1995. Plank on communi y
espi a ion in B an ield S ai (An a c ic Ocean) du ing aus al
sp ing. J. Plank on Res., 17 (8), 1647-1659.
Gómez e al. (1996) Modi ica ion o he elec on anspo sys em
(ETS) me hod o ou ine measu emen s o espi a o y a es o
zooplank on. S. A .J.Ma .Sci 17:15-20
Packa d, T.T. e al. 1996. Oxygen consump ion in he ma ine
bac e ium, Pseudomonas nau ica p edic ed om ETS ac i i y and
bisubs a e enzyme kine ics. J. Plank on. Res., 18 (9), 1819-1835.
Packa d, T.T. and Williams, P.L.B. 1981. Ra es o espi a o y
oxygen consump ion and elec on anspo in su ace seawa e
om he no hwes A lan ic Ocean. Oceanologica Ac a, 4, 351-
358.
Sa enko C. e al., 1995. Rela i e con ibu ion o
dehyd ogenases o espi a o y ETS ac i i y in some ma ine
o ganisms. Jou nal o Plank on Resea ch, 17(8),1593.