Plant Physiol.
(1982) 70, 42-445
0032-0889/82/70/0442/04/$00.50/0
Direct Spectrophotometric Measurement of Photosystem I and
Photosystem II Activities of Photosynthetic Membrane
Preparations from Cyanophora paradoxa, Phormidium laminosum,
and Spinach'
Received for publication January 26, 1982 and in revised form March 3, 1982
LEO P. VERNON AND STEPHAN CARDON
Department of Chemistry, Brigham Young University, Provo, Utah 84602
ABSTRACT
Vesicles prepared with the French press from membranes of cyanelles
of Cyanophora paradoxa retain 02 evolution activity with rates up to 500
micromoles 2,6-dichlorophenolindophenol reduced per hour per milligram
chlorophyll. This activity is immediately lost when the vesicles are trans-
ferred from the sucrose-phosphate-citrate preparation buffer into dilute
phosphate buffer. Similar preparations from Phormidium laminosum, a
thermophilic cyanobacterium retain activity under such conditions. Pho-
tosystem I activities of both cyanobacterial vesicle preparations were
determined by direct spectrophotometric measurement of N,N,N',N'- tet-
ramethyl-p-phenylenediamine photooxidation in the presence of anthraqui-
none-2-sulfonate. The rates so determined were compared with rates of 02
taken up in the presence of methyl viologen or anthraquinone-2-sulfonate
as electron acceptors. The predicted stoichiometry of two was observed for
moles of N,N,N',N'-tetramethyl-p-phenylenediamine oxidized per mole of
oxygen taken up. Anthraquinone-2-sulfonate was the betteF electron ac-
ceptor, and maximal rates of 943 micromoles per hour per milligram
chlorophyll for 02 uptake were observed for Phormidium laminosum prep-
arations in the presence of superoxide dismutase. For purposes of compar-
ison, spinach chloroplasts were assayed for similar activities. All prepara-
tions were readily assayed for photosystem I activity by the direct spectro-
photometric method, which has advantages of simplicity and freedom from
errors introduced by photoxidation of other substrates by photosystem I
when 02 uptake is measured.
Numerous redox agents have been utilized as artificial donors
and acceptors for both PSI and PSII of chloroplasts. These reac-
tions have been extensively reviewed by Hauska (4). PSII activity
is usually assayed spectrophotometrically by coupling the reduc-
tion of an optically absorbing agent to 02 evolution. A standard
measure of PSI activity involves the oxidation of donors such as
DPIP,2 DAD, or TMPD kept in the reduced form by excess
ascorbate. The oxidation of the ascorbate-donor couple is linked
to the reduction of an autooxidizable agent such as MV or a
2
Supported by a research grant from Brigham Young University.
Abbreviations: DPIP, 2,6-dichlorophenolindophenol; TMPD,
N,N,N',N'-tetramethyl-p-phenylenediamine; MV, methyl viologen; AQS,
anthraquinone-2-sulfonate; DAD, diaminodurene; DBMIB, 2,5-dibromo-
3-methyl-6-isopropyl-p-benzoquinone; SPC, a buffer (pH 7.0) containing
0.5 M sucrose, 0.5 M potassium phosphate, and 0.3 M sodium citrate; SOD,
superoxide dismutase.
442
quinone such as AQS (1 1). Such reactions are measured by means
of 02 uptake with an 02 electrode.
TMPD is readily reduced by PSII and oxidized by PSI, as
shown by the fact that it overcomes DBMIB inhibition of NADP
photoreduction coupled to water oxidation (12). In this case, it
serves as a bypass for electron flow past the DBMIB inhibition
site. In spinach chloroplasts, TMPD reacts with plastocyanin,
which serves as a direct donor to PSI (3). In this study we report
on the PSII activities of cyanobacterial preparations as well as the
direct spectrophotometric measurement of TMPD photooxidation
by PSI. The rates observed for PSI in this manner are compared
to the rates measured in the traditional way as 02 uptake with
excess ascorbate present. The impetus for the study was our
interest in the photoreactions of the cyanelle of Cyanophora
paradoxa, which although an endosymbiotic organelle, has the
photochemical capabilities of cyanobacteria. The lability of the
cyanelle PSII is shown as well as the ability of PSI to use ascorbate
itself as a donor to a significant extent. This is in contrast to other
photosynthetic systems in which ascorbate is a poor donor to PSI
(4).
MATERIALS AND METHODS
Spinach was purchased from a local market. The methods used
for growth of C. paradoxa and for isolation of the cyanelle were
those previously described (8). The original culture of Phormidium
laminosum (OH1-P), a gift from Dr. Richard Castenholz, was
grown as described by Jackson and Castenholz (5). Spinach chlo-
roplasts were prepared as previously reported (6) and finally
suspended in 0.4 M sorbital and 0.1 M Tris buffer (pH 7.0).
Photosynthetically active vesicles were prepared from cyanelles
and from P. laminosum cells by treatment in a French Press at
14,000 p.s.i. in the medium described by Dilworth and Gantt (12)
which contained 0.5 M sucrose, 0.5 M K-phosphate and 0.3 M
sodium citrate, all at pH 7.0. This is designated SPC buffer. Serum
albumin was not added, as was previously done by Katoh and
Gantt (7). Better activity was observed for PSII if the final vesicle
preparation contained 0.2 mg Chl/ml or more.
PSII was assayed by DPIP photoreduction in a medium pre-
pared with 0.5 ml SPC buffer, 30 ,tl 2.0 mm DPIP, and 10 ,ul of the
preparation to be tested. PSI activity was measured spectropho-
tometrically in 0.6 ml of a medium consisting of 0.5 ml SPC
buffer, 0.67 mm TMPD, 3.3 mm KCN, 0.67 mm MV or 0.83 mM
AQS, and 20 ,ul of the preparation to be tested. The stock solution
of 20 mm TMPD was in methanol and was kept at 0°C in the
dark to prevent oxidation. As the TMPD became oxidized in this
stock solution, the background rate of TMPD oxidation in the
assay became greater. New solutions were prepared weekly. The
 SPECTROPHOTOMETRIC MEASUREMENT OF PSI
original cholorplast or cyanobacterial preparation was usually
diluted 1 to 10 in SPC buffer for the assay. When DCMU was
present, it was at 0.1 mm. For the determination of PSI activity by
means of 02 uptake, the assay mixture contained 1.3 ml SPC
buffer, 4.0 mm NH4Cl, 1.0 mm KCN, 0.1 mm MV or AQS, 0.1 mM
TMPD, and 2.0 mm ascorbate in a final volume of 2.6 ml. This
reaction was measured with a Yellow Springs Instrument Co. Ox2
electrode immersed in the reaction mixture which was stirred with
a magnetic stirrer and maintained at 2°C with a water bath.
Illumination was with a 150-w tungsten lamp augmented with the
illuminator described below for the spectrophotometric assay.
Light intensity on the reaction vessel was 1 x 106 ergs/cm2. s.
Spectrophotometric assays were performed in a DU 8 Beckman
spectrophotometer which had been modified to accommodate an
illumination system consisting of a model 170D illuminator (Do-
lan-Jenner Co., Woburn, MA) which transferred light to the
reaction cuvette through a light pipe of one-half inches diameter.
The incident light was passed through a red cutoff filter (Coming
2-58) and a blue cutoff filter (Coming 4-96) was placed between
the reaction cuvette and the housing containing the photocell. A
1-ml cuvette was used to allow the entire reaction mixture to be
illuminated, otherwise local differences in absorbance were present
which led to spurious results. Light intensity at the reaction vessel
was 5 x 105 ergs/cm2. S which was saturating. Both reactions were
determined at 590 nm, which is close to the absorption peak of
DPIP, and is located at the small trough between the two major
absorption peaks of TMPD. The specific millimolar absorptivities
of DPIP and TMPD used for calculation of rates of reaction were
16 (10) and 8.04/cm, respectively.
RESULTS AND DISCUSSION
In our experiments involving the photochemical apparatus of
the cyanelle of C. paradoxa, it was necessary to measure both
photosystem activities independently. Once it was determined that
preparation of cyanelle vesicles by use of the French Press with
SPC buffer (1) produced an active preparation for 02 evolution,
as evidenced by DPIP photoreduction, it became possible to study
both photosystems. Preliminary experiments designed to follow
PSI activity by a direct spectrophotometric measurement of
TMPD oxidation proved promising, and this reaction was used
routinely, not only with cyanelle preparations, but with another
cyanobacterium and with spinach chloroplasts.
E
jl_r
so
A was extended by Dilworth and Gantt (1) with the red alga
0
0).36
ILO
w retention of the phycobilisomes on the membrane surface, indi-
0
z
.24
m formation leading to inactivity.
z .12
CD
CD off
30 60 go 30
TIME (seconds)
FIG. 1. Spectrophotometric measurement of PSI and PSII activities.
Vesicles of C. paradoxa and spinach chloroplasts were prepared from the
cyanelles as described in "Materials and Methods." A, DPIP photore-
duction as a measure of PSII. The PSII assay system contained 6.1 jig Chl
a in cyanelle vesicles. B, TMPD photooxidation as a measure of PSI. The
PSI assay system contained AQS as the electron acceptor and 3.0 ,ug Chl
a from spinach chloroplasts. DCMU was present as indicated as 10' M.
The rates of reaction in Mmol/h mg Chl were 498 for DPIP photoreduc-
tion, and 1,532 and 1,848 for TMPD photooxidation, the latter in the
presence of DCMU.
443
Table I. Instability of 02 Evolution in Photosynthetic Membrane Vesicles
Preparedfrom Cyanelles of Cyanophora paradoxa
The assay system for PSII activity coupled to DPIP photoreduction is
given under "Materials and Methods." Where indicated, phosphate buffer
(pH 7.0) of 0.025 ionic strength was substituted for the sucrose phos-
phate:citrate (SPC) buffer in the assay mixture.
H20-- DPIP Activity
Conditions
Cyanelles P. laminosum Spinach
,imol/mg Chl.h
SPC buffer 390 204 330
Dilute phosphate buffer 5 185 256
Dilute phosphate buffer
after 10 min 0 172 205
Figure 1 shows the absorbance changes that occur with a
cyanelle preparation at 590 nm with either DPIP (measuring PSII)
or TMPD (measuring PSI). For the PSI reaction, an autooxidiz-
able reagent such as MV or AQS must be present to accept
electrons from PSI. As the TMPD photooxidation reaction pro-
ceeds, it departs from linearity as the oxidized form accumulates,
most likely due to the ability of the oxidized form to serve as an
acceptor for PSII. The addition of DCMU to the reaction mixture
extends the period of linearity and generally causes an increase in
the observed rate of TMPD photooxidation. As shown below, this
measure of PSI activity is a valid measurement, as determined by
a comparison with the more traditional method of measuring 02
uptake in a similar system, in which excess ascorbate is present to
keep TPMD in the reduced form. The spectrophotometric assay
is particularly easy to perform with the Beckman DU 8 spectro-
photometer, inasmuch as absorbance decreases (DPIP) or in-
creases (TMPD) can be followed using the same apparatus and
same program at 590 nm. There is a slow oxidation of TMPD in
the dark, as evidenced by the slow reaction before the light is
turned on (Fig. 1). The initial dark reaction can be minimized by
the addition of cyanide and utilization of freshly prepared TMPD
solutions.
It is known that PSII from cyanobacteria is difficult to isolate
in an active form, and it was the earlier work of Katoh and Gantt
(7) which showed that use of the SPC buffer, containing high
concentrations of sucrose, phosphate, and citrate, allowed the
isolation of active vesicles from Anabaena variabilis. This work
Porphyridium cruentum, in which they demonstrated a stable active
preparation from this organism, showing rates of about 450 ,tmol/
h .mg Chl for 02 evolution. The key to retention of activity is the
cating that the phycobilisomes are intimately connected to the 02
evolving system, and their removal allows changes in PSII con-
By use of the Dilworth and Gantt procedure, active preparations
are routinely prepared from the cyanelle of C. paradoxa, showing
that the same considerations apply in this case. The preparations
retain activity for several days in SPC buffer in the cold (2°C),
although it does decrease slowly. Table I shows that the PSII
activity of cyanelle preparations is lost immediately if DPIP
photoreaction is assayed in dilute phosphate buffer. For purposes
of comparison, the thermophilic cyanobacterium P. laminosum
was subjected to a similar treatment. This cyanobacterium is much
more stable in terms of its 02 evolution activity, and the data of
Table I illustrate that this stability is reflected in preparations
from this cyanobacterium being able to perform DPIP photore-
duction in a reaction mixture with dilute buffer. We have not
performed detailed experiments to detect the removal of phyco-
bilisomes from the vesicle preparations in dilute buffer, but cen-
trifugation of vesicle preparation after dilution with the original
444 VERNON AND CARDON Plant Physiol. Vol. 70, 1982
Table II. Photochemical Activities of Spinach Chloroplast and Cyanobacterial Membrane Preparations
PSI activities for TMPD photooxidation were measured either directly by spectrophotometric determination of TMPD photooxidation or by the 02
electrode in the presence of ascorbate and either MV or AQS. Rates are expressed as pmol/h.mg Chl of TMPD oxidation or 02 uptake. Where
indicated, 0.1 mg SOD was present and DCMU was 0.1 mM. When SOD was present, KCN was omitted. The ratios given for the two methods for
measuring PSI activity are calculated after substracting the rate of 02 taken up in the absence of TMPD, which is a measure of ascorbate oxidation by
PSI.
Rates of TMPD Photooxidation
Preparation
H20 DPIP TMPD -* MV TMPD --AQS
(1) (2) (3) (4) (5)
Spectro Electrode (2)/(3) Spectro Electrode (4)/(5)
pmol/mg Chl.h ratio umol/mg Chl.h ratio
Spinach chloroplasts 274 1,452 912 1.67 1,911 942 2.12
+DCMU 0 1,823 881 2.17 2,036 935 2.28
+SOD 302 1,351 779 1.84 1,893 978 2.03
+DCMU, SOD 0 1,307 680 2.09 1,820 848 2.26
-TMPD + DCMU, SOD 0 43
Cyanelle 390 678 608 1.71 1,295 762 2.35
+DCMU 0 640 585 1.63 962 691 2.00
+SOD 397 579 570 1.62 757 621 1.92
+DCMU, SOD 0 540 550 1.70 687 611 1.82
-TMPD + DCMU, SOD 0 226
P. laminosum 330 1,423 1,024 1.42 2,490 1,264 2.01
+DCMU 0 1,619 728 2.30 2,605 1,243 2.13
+SOD 370 1,247 653 1.99 2,079 997 2.14
+DCMU, SOD 0 1,118 557 2.10 2,005 943 2.18
-TMPD + DCMU, SOD 0 25

SPC buffer solution and after dilution with dilute phosphate
buffer shows that almost all of the phycocyanin from the cyanelle
preparation is recovered in the supernatant fraction after dilution
with phosphate buffer, and less than half the phycocyanin is
recovered from the supernatant of the suspension diluted with
SPC buffer. By contrast, much less of the phycocyanin is recovered
in either supernatant when P. laminosum vesicles are subjected to
the same treatment. This agrees with the more detailed analysis
performed by Dilworth and Gantt (1).
The direct spectrophotometric measurement of TMPD pho-
tooxidation is simple to perform, but needs to be compared to the
generally accepted assay involving 02 uptake in the presence of
excess ascorbate. Table II presents data obtained from three
materials, comparing both reactions with either MV or AQS added
as the autooxidizable acceptor for PSI. The rates reported are for
TMPD oxidation directly or for 02 uptake, in terms of ,umol/h-
mg Chl. Since 02 is taken up to form H202, as shown by Ort and
Izawa (11), there are two electrons transferred per 02 in this
reaction compared to the one electron involved in the photooxi-
dation in TMPD, which is oxidized to a stable semiquinone known
as Wurster's blue (9). So long as initial rates are taken for TMPD
oxidation, the one electron step is valid. As shown in Table II,
with some variation, the expected stoichiometry of 2 mol TMPD
photooxidized to each mol of 02 consumed is realized for all three
preparations, showing the equivalence of the two methods of
measuring PSI activities.
Some features of the data of Table II are worthy of note.
Addition of DCMU to the spectrophotometric assay causes an
increase in the rate of reaction, which may reflect the ability of
the oxidized form of TMPD to serve as an electron acceptor for
PSII. When DCMU is added to the 02 electrode assay, a decrease
in activity is noted, which indicates that some electrons are flowing
through PSII, either from water oxidation or oxidation of the
added donor, either TMPD or ascorbate. For all three prepara-
tions, the rates observed with AQS as the electron acceptor were
greater than those for MV. This was especially true for P. lami-
nosum, for which the AQS-supported reaction was almost twice
that observed with MV.
The reactions observed with cyanelle vesicles differ in two
significant ways. They are the lowest of the rates observed, and
there is appreciable photooxidation of ascorbate in the absence of
TMPD. Since DCMU is present in the assay mixture, the ascorbate
must be oxidized by PSI. This is in contrast to most photosynthetic
systems examined to date (2, 4).
The enzyme SOD affects both photoreactions studied. Its ad-
dition causes a slight stimulation of DPIP photoreduction, and a
more significant inhibition of 02 uptake. The nature of the stim-
ulation of DPIP photoreduction is not known, but the effect on
02 uptake by ascorbate-supported PSI reactions has been studied
by Ort and Izawa (11). In the absence of the enzyme, the super-
oxide ion generated in the oxidation of the reduced acceptor, MV
and AQS, interacts with ascorbate and is reduced to peroxide by
ascorbate. This increases the amount of 02 taken up by the
electrons flowing through PSI, and changes the stoichiometry of
the reaction. The addition of SOD to reactions geared for 02
uptake decreases the amount of 02 taken up, and leads to the
correct balance of 02 uptake per electron flowing through PSI.
The fact that SOD also inhibits the oxidation of TMPD measured
directly indicates that superoxide ion can interact with and oxidize
TMPD in such reaction mixtures, also leading to a high value of
TMPD oxidation.
The experiments outlined in Table II are directed toward PSI
reactions, and the DPIP activities of these particular preparations
were somewhat low. The rates of DPIP photoreduction by cyanelle
vesicles range up to 500 ,umol/h.mg Chl, (Fig. 1). The maximal
rate observed for P. laminosum vesicles is in the same range, 481
,umol/h -mg Chl.
The direct spectrophotometric assay for TPMD photooxidation
by PSI is shown to be a valid assay. It is also an easy assay to
perform in conjunction with the standard DPIP photoreduction
assay for PSII activity. The 02 uptake assays for PSI in the
presence of excess ascorbate are also easy to perform, but as shown
 SPECTROPHOTOMETRIC MEASUREMENT OF PSI
in Table II, one must be sure that ascorbate itself is not contrib-
uting to 02 uptake in a major way which can produce erroneous
results, as was observed with cyanelle vesicles. In the case of the
cyanelle, we feel that the direct spectrophotometric assay is the
more accurate assay, inasmuch as it does not have the potential
complications of other electron donors supplying electrons to PSI,
and so long as the initial rates of TMPD photooxidation are linear,
a reliable rate for this activity can be obtained.
The rates of PSI activities reported here are in agreement with
those reported by others. Ort and Izawa report rates for spinach
chloroplast (11) of 775 for diaminotoluene and 1,250 for diami-
nodurene, while Hauska et al. (2) report rates of 860 for TMPD,
890 for diaminodurene, 1,880 for a mixture of TMPD and dia-
minodurene, 450 for DPIP, and 2,100 for a mixture of TMPD and
DPIP. All these rates are for 02 uptake to the peroxide level, or
two electrons per 02 molecule.
A direct spectrophotometric oxidation of DPIP can also be
observed to some extent, but the observed rate is not linear (data
not shown here). This reflects the fact that DPIP is a better
electron acceptor for PSII reactions than is TMPD but is not as
good a donor to PSI.
The expected stoichiometry of 2 mol TMPD photooxidized/
mol of 02 taken up is generally realized, but with spinach and P.
laminosum the ratios are somewhat above 2, and for cyanelles the
ratio is below 2. There may still be factors relating to photooxi-
dation reactions of PSI that are not immediately apparent. The
situation with cyanelles is complicated by the high rate of ascor-
bate photooxidation by PSI. With spinach and P. laminosum the
rates are close enough to allow either method to be used in
comparative measurements of PSI activity. In either case DCMU
445
and SOD must be added to ensure that the TMPD oxidized or 02
taken up accurately measure PSI activity.
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