'aurnal of Chemical Ecology. Vol. 22. No. I0.

/996

PHLOROTANNIN-PROTEIN INTERACTIONS

J. L E W I S STERN, I ANN E. H A G E R M A N , ~-'*

PETER D. STEINBERG, 3 and PAMELA K. MASON 4

'Department of Biology, University of New South Wales

Kensington, New South Wales, Australia 2033
"-Department of Chemistry, Miami Universi O'
Oxford, Ohio 45056
3Department of Biology. University of New South Wales
Kensington, New South Wales. Australia 2033
aDepartment of Ct~emistry, Miami University
O~Jbmt. Ohio 45056

(Received December 18. 1995; accepted May 19. 19961

Abstract--Tannins arc one of the most broadly distributed types of plant

secondary compounds, and have been the t't~-al point Ik~r many studies of
plant/herbiw~re interactions. Tannins interact strongly with proteins, so that
the fate and effects of ingested tannins are in parl dependent on the mode of
interaction of the tannin with dietary and endogenous proteins in an herbi-
vore's gut. We investigated the factors affecting the precipitation of proteins
by phlorotannins I'rom three species t~l" marine brown algae: C(lrpophylhttn
maschalot'at'pltm. Eckhmia mldiata, and Lob~q~hora (,ariegat,. Phlorotannins
were precipitated by proteins in a pH-dependent and concentration-dependent
fashion. Precipitation als~ varied as a function of the presence of reducing
agent, the type of phiomtannin or protein used. and the presence of organic
solvents such as hydr~)gen bond inhibitors. Of particular significance was the
ability of sru'ne phlorotannins to oxidize and form covalent bonds with sru~e
proteins. In contrast, under similar experimental conditions three types of
terrestrial tannins (procyanidins. profiselinidins, and gallotannins) apparently
did not fiwm Covalent complexes with proteins. Our results suggest several
ways in which the biological activity of phlomtannins may vary as a function
of the properties of the gut environment of marine herbivores. Moreover, we
identify specific structural characteristics of phlomtannins which affect their
tendency tt~ oxidize, and thus. their potential effects on marine herbivores.

To whom correspondence should be addressed.

1877

II(P~ (1331/q~iI(KK) 1~7754Y4 5(I/(I Iqg6 I'lcnum Pubh,hillg (',

1878 STERN ET AL.

Key Words--Tannin-protein interaction, tannins, hydrogen bonding, protein

precipitation, marine brown algae, phlomtannins, Carpophyllum maschalo-
carpum, Eckhmia radiata, Lohophora variegata.

INTRODUCTION

Tannins, or polymeric phenolics, are one of the most broadly distributed types
of plant secondary metabolites in terrestrial (Harborne, 1991) and marine (Ragan
and Glombitza, 1986) plants. Tannins have important effects on both marine
and terrestrial herbivores, but there is substantial variation in the biological
activities of tannins against different herbivores (Bemays et al., 1989; Steinberg,
1993: Appel, 1993: Boettcher and Targett, 1993). In part this variation is because
tannins do not have a single biological activity, but may act as feeding deterrents
(Steinberg, 1988; Clausen et al., 1990); digestibility reducing agents (Robbins
et al., 1987~ Boettcher and Targett, 1993): or toxins (Bemays et al., 1980),
The variation in biological activity of tannins is due in pan to variation in
tannin structure (Zucker, 1983). For example, terrestrial tannins bind proteins
selectively (Hagerman and Butler, 1981), and some mammals utilize that selec-
tivity by secreting salivary tannin-binding proteins which neutralize specific
tannins (Hagemaan and Robbins, 1993). Even minor structural variation, such
as stereoisomerism, affects the activity of some terrestrial proanthocyanidins as
feeding deterrents (Clausen et al., 1990), The ability of phlorotannins to inhibit
protein assimilation by an herbivorous fish varied with the molecular weight of
the tannin (Boettcher and Targett, 1993). Phlorotannins from different species
of brown algae also varied in their activity as feeding deterrents for marine
herbivores (Steinberg and van Altena, 1992: van Altena and Steinberg, 1992)
although the structural elements leading to the variation in activity were not
identified.
The variation in the biological activity of tannins can also be attributed to
variations in the gut physiology and biochemistry of various herbivores. For
example, the ability of tannins to precipitate proteins is dependent on pH, the
presence of surfactants, and the concentration of metal ions (Hagerman and
Butler, 1978; Martin and Martin, 1984: Tugwell and Branch, 1992). All of
these factors a ~ c t how tannins affect herbivores, and how herbivores can min-
imize the detrimental effects of tannins on ecological or evolutionary time scales
(Bernays et al., 1989: McArthur et al., 1991).
The ability of tannins to precipitate protein has been examined in many
studies. However, with few exceptions (Tugwell and Branch, 1992; Boettcher
and Targett, 1993), most of the work on interactions between proteins and
tannins has been done using terrestrial tannins (Hagerman, 1992). It may be
inappropriate to draw conclusions about marine herbivore/phlorotannin inter-
actions based on knowledge about terrestrial tannins. Phlorotannins (1-4, Figure
PHLOROTANNIN-PROTEIN INTERACTIONS 1879

1), which are known only from the Phaeophyta (brown algae) (Ragan and Glom-
bitza, 1986), are structurally quite unlike terrestrial tannins (5, 6, Figure 1).
Furthermore, the gut morphology, physiology, and biochemistry of marine her-
bivores are quite unlike those of terrestrial herbivores (Horn, 1989). We have
therefore conducted experiments designed to define the parameters that control

OH OH

o,OvOH
HO OH OH HO OH
OH O OH

1 2 ~OH
HO

OH
0 OH HO~ T / O H )H
HO

3 O..~OH 4 O~o~"~"~OH
HO

OG O OH

HO O ~OH
~ y "~ '"OH
OH HO HH~~o O~ } ~ , ~ OH
G= OH G>G = HO OH
OH O O

E ''"
HO OH
OH
5 6
FIG. 1. Structural formulae of various tannins. 1, tetrafuhalol A, is a fuhalol-type phlo-
rotannin; 2, hydroxytetraphlorethol, is a hydroxyphlorethol-type phlorotannin; 3, tetra-
phlorethol C, is a phlorethol-type phlorotannin; 4, tetrafucol A, is a fucol-type
phlorotannin; 5, heptagalloyl glucose, is a gallotannin; 6, epicatechin-413 ~ 8-catechin
(procyanidin B-I), is a procyanidin-type condensed tannin.
1880 STERN ET AL.

precipitation of phlorotannins by proteins. Phlorotannins from three species of

brown algae and several proteins were used to evaluate structure-activity rela-
tionships. We examined the effects of pH, of hydrogen bonding solvents, and
of detergents and hydrophobic solvents on the interaction between phlorotannins
and proteins.
In the course of our study, it became clear that oxidation reactions were
an important feature of phlorotannin chemistry. It has been postulated that oxi-
dation of tannins may be crucial to their biological activity (Appel, 1993), and
it has been demonstrated that tannic acid precipitates protein more effectively
in the presence of oxidants (Leatham et al., 1980). However, we believe that
our work with phlorotannins is the first clear demonstration of a role for spon-
taneous oxidation in the formation of tannin-protein complexes. We were unsuc-
cessful in our attempt to demonstrate similar reactions for several representative
terrestrial tannins.

METHODS AND MATERIALS

Reagents
Phlorotannins were purified from Ecklonia radiata (Order Laminariales)
and Carpophyllum maschalocarpum (Order Fucales) using methods developed
by Ragan and Glombitza (1986) as modified by Steinberg and van Altena (1992).
High ( > 100,000 ainu) and moderate (5,000-100,000 amu) molecular weight
phlorotannins from b~bophora variegata COrder Dictyotales) (Boettcher and
Targett, 1993) were provided by Nancy M. Targett (University of Delaware,
Lewes, Delaware, 1995). Terrestrial tannins were purified from Sorghum grain,
from commercial tannic acid or from crude commercial quebracho tannin (Hag-
erman and Butler, 1980b; Asquith and Butler, 1985; Hagennan and Klucher,
1986). Phlorotannin preparations were comprised of polydisperse tannins with
molecular weights ranging from 6,000-100,000 (Van Altena and Steinberg,
1992; Boettcher and Targett, 1993). Condensed tannins from Sorghum grain or
quebracho did not contain any low molecular weight phenolics (Hagerman and
Butler, 1980b). Purified tannic acid is a mixture of polygalloyl esters ranging
from pentagalloyl glucose to decagalloyl glucose (Hagerman and Klucher, 1986).
Proteins and 2,4-dimethoxybenzaldehyde (DMBA) were obtained from
Sigma Chemical Co., St. Louis, MO. Radiolabeled bovine serum albumin was
prepared by reacting Na~251 with bovine serum albumin in the presence of Chlor-
amine T (Hagerman and Butler, 1980a). The iodinated protein was dialyzed
either against Buffer J or against Buffer J containing 15 mM DTT (dithiothreitol)
prior to conducting precipitation assays. Protein compositions, isoelectric points,
and molecular weights were obtained from the literature (Sugiyama et al., 1968;
pH L O R O T A N N[ N-PROTI~I N I N T E R A C T I O N S 1881

Mahler and Cordes, 1971; Peters, 1975; Malmud and Drysdale, 1978; Paech,
1985).
Phlorotannin solutions (10 #g/t~L) were prepared daily in methanol. Pro-
teins were dissolved in 0.11 M sodium chloride and stored at 4C. Most proteins
were prepared as 10/~g//zL solutions, but ribulose-l,5-bisphosphate carboxyl-
ase/oxygenase (RuBPC) was prepared as a saturated solution (approximately 2.5
/~g/p.L) and centrifuged to yield a clear solution.
Acidified acetic acid was prepared by mixing concentrated hydrochloric
acid with glacial acetic acid (16 mL concentrated hydrochloric acid per 100 mL
solution). Fresh solutions of DMBA (2 g/100 mL glacial acetic acid) were
prepared fresh daily; this solution turned purple with age (Stern et al., 1996).
For the standard precipitation reactions at pH 5.0, Buffer J was prepared
(0.1 M acetate, 0.43 M sodium chloride, pH 5.0) and stored at 4C. For pH
profiles, 0.1 M sodium phosphate buffers were prepared at various pH values
between 2 and 12. A stock 1 M solution of the reducing agent DTT in water
was prepared and stored at - 2 0 C . Buffers were brought to a final concentration
of 15 mM DTT daily by adding an appropriate amount of the stock solution of
DTT.

Phlorotannin-Protein Precipitation Assay

We used the new DMBA assay, described more fully in Stern et al. (1996),
to measure phlorotannins after they were precipitated by protein. Buffer (180
#L) was mixed with the phlorotannin solution (10/zL of 10 Izg/p.L phlorotannin)
in conical glass centrifuge tubes (12-15 mL capacity). Protein (20/~L of 10 p.g/
p.L protein) was added and the samples were mixed. The samples were incubated
for 30 min at room temperature, and were then centrifuged at 2000 g for 15
rain. The supernatants were removed by aspiration and the surfaces of the pellets
were gently washed with 100/xL buffer. The samples were centrifuged again
t'or 5 rain, and the supernatants removed by aspiration.
In order to quantitate the phlorotannin in the precipitates, it was necessary
to redissolve the precipitated complexes. In the initial experiments with protein
and phlorotannim the protein-phlorotannin precipitates were dissolved directly
in glacial acetic acid containing DMBA and hydrochloric acid. Although this
was successful for preliminary experiments with Ecklonia tannin and bovine
serum albumin, as we began to extend the work we found that in some cases
the protein-phlorotannin precipitate did not completely dissolve in this reagent.
We unsuccessfully tested a variety of conditions such as extremes of pH or
addition of protein denaturants for dissolving the precipitates. We found that for
most proteins the complex could be completely dissolved by vigorous agitation
of the precipitate in 10 #L of N,N-dimethylformamide. This procedure was used
in all subsequent assays.
1882 STERN ET AL.

Immediately after dissolving the pellets, 1.25 mL of the acidified acetic

acid was added to each sample. It was necessary to add the acidified glacial
acetic acid reagent immediately after dissolving the precipitate in the dimethyl-
formamide to prevent destruction of the phlorotannin. At timed intervals, 1.25
mL of DMBA reagent was added to each sample; the samples were immediately
mixed and placed in a water bath at 30C. Exactly 60 min later, the absorbance
of each sample was determined at 510 nm (Stem et al., 1996).
A small amount of phlorotannin adsorbed to the tubes, resulting in back-
ground absorbance. To correct for that background, a sample containing phlo-
rotannin and buffer but no protein was treated like the other samples and its
absorbance (typically about 0.06) was subtracted from the absorbance for each
sample. The total amount of phlorotannin used in each experiment was deter-
mined by reacting 10 p.L of the phlorotannin solution, 10 #L N,N-dimethylfor-
mamide and 1.25 mL of the acidified acetic acid with 1.25 mL of DMBA reagent
for exactly 60 min at 30C. The amount of phlorotannin precipitated was cal-
culated as a percent of the total amount of phlorotannin.

Precipitation of 1251Bovine Serum Albumin

This assay was used to determine the amount of protein precipitated by the
various phlorotannins. In this assay, the amount of radiolabeled protein precip-
itated is determined by direct radiochemical counting. Since it is not necessary
to dissolve the tannin-protein complex to count the radiolabeled protein, this
assay was particularly useful for quantitating insoluble covalent complexes
obtained with phlorotannins.
Tannins were dissolved in methanol and 100 #L was added to 300 #L of
the appropriate buffer containing 50/zg of iodinated protein. The concentration
of the tannin in the solution was set so that not all of the protein precipitated.
After 15 rain the samples were centrifuged, the superuatants were removed and
the precipitates were gently washed with 100 #L of the appropriate buffer. The
samples were centrifuged again, the supernatants were removed and the precip-
itates were counted with a gamma counter.

RESULTS

Precipitation of Phlorotannin by Protein

In order to determine how biologically relevant parameters affect the inter-
action between phlorotannins and protein, it was first necessary to optimize the
conditions for precipitation. We evaluated the effects of time, temperature, and
reactant concentrations on precipitation of phlorotannins by proteins. Precipi-
tation of Ecklonia phlorotannins by protein occurred rapidly (Figure 2a). The
PHLOROTANNIN-PROTEI N INTERACTIONS 1883

amount of phlorotannins precipitated was similar for incubation times ranging

from 15 rain to 24 hr, and at temperatures ranging from 4C to 35C. Precip-
itation of Carpophyllum phlorotannins increased slowly with time when the
reducing agent DTT was present in the incubation mixture (Figure 2b). We
routinely incubated phlorotannins and protein for 30 rain at room temperature
before analyzing the precipitate.
The composition of the phlorotannin-protein precipitate was dependent on
both the amount of protein and phlorotannins present. When the protein-to-
phlorotannin mass ratio was small ( < 2 : 1), between 60 and 100% of the phlo-
rotannins were precipitated and all of the protein was precipitated. The efficiency
of phlorotannin precipitation depended on the phlorotannins, the protein and the
buffer used. If the protein-to-phlorotannin ratio was large ( > 5 : 1), less than
60% of the phlorotannins were precipitated, and only a fraction of the protein
was precipitated. The mass ratio of protein-to-phlorotannin was routinely set at
2 : 1 to maximize the amount of phlorotannin that was precipitated.

Effects of Reducing Agents

We found that even dimethylfbrmamide did not completely dissolve pre-
cipitates formed by Carpophyllum phlorotannins and certain proteins especially
when the protein-to-phlorotannin ratio was small, Precipitates comprised of Car-
pophyllum phlorotannins and trypsin, chymotrypsin, or cytochrome c were dark-
colored and resistant to dissolution. Ecklonia phlorotannins and cytochrome c
also formed dark-colored precipitates which were difficult to redissolve. The
color of these complexes suggested that oxidation reactions were involved, and
the resistance to dissolution suggested covalent bonds had formed between the
tannin and the protein. We used reducing agents to evaluate the role of oxidation.
When the phlorotannins were mixed with any of these proteins in buffer con-
taining high concentrations of reducing agents, such as 50 mM/3-mercaptoeth-
anol or 15 mM DTT, the complexes which formed were pale colored and could
usually be dissolved in dimethylformamide, This indicated the importance of
oxidation in phlorotannin-protein interactions, so we subsequently routinely
added 15 mM DTT to the incubation buffer.
The addition of reducing agents also had the benefit of maximizing the
color yield obtained in the DMBA assay. For example, when DTT was omitted
from the reaction mixtures, less Carpophyflum phlorotannins were detected in
precipitates formed during long (6-24 hr) incubations with protein than were
detected after short (30 min) incubations (Figure 2b). In the presence of reducing
agent, the amount of precipitated Carpophyltum phlorotannins increased with
long incubations. It is likely that during the long incubation without reducing
agent the phlorotannins spontaneously oxidized, decreasing their reactivity with
DMBA. We minimized oxidative interference with the DMBA assay by incor-
1884 STERN ET AL

140
a
"(3
(1)
120
9- 100
o
I--

80
c A4C
c
c
0 60
O
[] 2 7 C
I--
O 40
t- c, 3 5 C
20
o"9- Ecklonia
0 l I

0 5 10 15 20 25
time (h)

140
"10
--" 1 2 0 /

-~ 100
(1)
t.-

" 80
c
c
c 60
o DTT
~
0 40
l-
o no DTT
~ 20
Carpophyllum
0
0 5 10 15 20 25
time (h)
pH I.OROTA N NI N-PROTEIN INTERACTIONS 1885

porating the reducing agent DTT in the precipitation step and by maintaining
the redissolved samples at low pH, since phenolic oxidation occurs less readily
at lower pH values (Ronlan, 1978).

Composition of Phlorotannin-Protein Complexes

For all proteins tested, the amount of either Carpophyttum or Ecklonia
phlorotannins detected in the precipitate was increased by the addition of the
reducing agent DTT to the incubation buffer (Figure 3). Further experiments
with phlorotannins from Lobophora indicated that the magnitude of the effect
of DTT was both protein- and phlorotannin-specific, For example, precipitation
of high molecular weight l_z~bophora phlorotannins by RuBPC or pepsin was
not affected by DTT, but precipitation by bovine serum albumin, lysozyme, or
trypsin was DTT-dependent (Figure 3). The amount of low molecular weight
Lz)bophom phlorotannins precipitated was not affected by addition of DTT for
any protein tested (Figure 3).
We used radiolabeled bovine serum albumin to confirm the effect of reduc-
ing agents on phlorotannin precipitation. We determined the amount of protein
precipitated by phlorotannins or terrestrial tannins in the presence and absence
of reducing agents. The amount of protein precipitated by Carpophyllum or by
high molecular weight l~)bophora phlorotannins was increased by addition of
DTT to the incubation mixture (Table 1), The reducing agent did not affect the
amount of protein precipitated by Ecklonia or low molecular weight l~bophora
phlorotannins, or by the three terrestrial tannins, Not only was precipitation of
tannic acid, a terrestrial gallotannin, insensitive to DTT (Table 2), but we found
that fairly vigorous conditions wcre required to oxidize gallotannins. We were
able to oxidize gallotannins only at pH values above 8 in the presence of an
oxidant (A. E. Hagerman and S. Y. Harper-Avilla unpublished data), In con-
trast, we have found that some phlorotannins are susceptible to oxidation by air
in slightly acidic to neutral solutions.
The activity of the reducing agent was pH-dependent (Figure 4). In general,

FIG, 2. Kinetics of phlorotannin precipitation by bovine serum albumin. Phlorotannin

(10 ~g) was mixed with protein (20 #g) in pH 5 phosphate buffer, After the indicated
incubations~ mixtures were centrifuged, and the supematants aspirated off. The pellets
were redissolved in acidified acetic acid and reacted with DMBA in acetic acid at 30C
for exactly 60 rain betbre the absorbance at 510 nm was determined. Each point repre-
sents the mean of three determinations and the error bars represent standard deviations.
a, Precipitation of Eckhmia phlorotannin at 4C, 27C, or 35C. b, Precipitation of
Carpophyllum phlorotannin at 25C in the pre,~nce or absence of DTT, a reducing
agent.
1886 STERN ET AL.

100 . . . . . 100 . . . . . . .
Ecklonia ' !without DTI- Carpophyllum [ ]without D'I-I"
80 .. with D'I-I" ) .0 with DTT

~. 60 '= 60 . . . . .
c . * .g

cc 40 - . Q 40
m ro

z 20 5 20 ......
I
0 0
BSA LYSO TRY RuBPC PEP BSA LYSO TRY RuBPC PEP

100 100 --
I Lobophora > 100 iwilhout DTT i Lobophora <100 I ]without 01"3-
.th o ~ "~ mwith o]-r

~o ~ 20

0
BSA LYSO TRY
. .
RuBPC
. '11
.
PEP
~ 0 "
BSA
!J
LYSO
-J.
TRY
:L,
RuBPC PEP

FIG. 3. Precipitation of phlorotannin in the presence and absence of DTT, a reducing

agent. The standard D M B A method was used, but D T T was omitted from buffer J for
reactions without DTT. Each bar represents the mean of three replicates. Significantly
different values (t-test) between treatments with and without D T T are indicated ( * * for
P < .0], * f o r P < .05).

addition of DTT had little effect on the composition of protein-phlorotannin

complexes formed at low pH. DTT increased the amount of phlorotannin pre-
cipitated at slightly acidic, neutral, or basic pH values.

Phlorotannin-Protein Interactions
To evaluate interactions between unoxidized phlorotannins and proteins,
we examined the effects of pH and of various solvents on precipitation in the
presence of reducing agents. The precipitation of phlorotannins by various pro-
teins is strongly pH-dependent (Figure 4; Figure 5). The shape of the pH profile
is largely dictated by the characteristics of the protein. The pH range which
favored precipitation was related to the acidity or basicity of the protein. For
example, the very acidic protein pepsin (pl = 2.2) was precipitated only in
acidic solutions (pH < 6). The slightly acidic proteins bovine serum albumin
(pI = 4.9) and RuBPC (pI = 4.9) were precipitated in slightly acidic to neutral
PHLOROTANNIN-PROTEIN INTERACTIONS 1887

TABLE I. PRECIPITATION OF RADIOLABELED BOVINE SERUM ALBUMIN BY VARIOUS

TANNINS IN THE PRESENCE OR ABSENCE OF DTT. THE AMOUNT OF 125/-BOVINE SERUM
ALBUMIN IN THE TANNIN-PROTEIN PRECIPITATE WAS DETERMINED BY GAMMA
COUNTING. TO ENSURE THAT CHANGES IN THE AMOUNT OF PROTEIN PRECIPITATED
COULD BE DETECTED, THE PROTEIN-TO-TANNIN MASS RATIO WAS SET SO THAT THERE
WAS ALWAYS SOME UNPRECIPITATED PROTEIN LEFT IN THE REACTION MIXTURE. THE
VALUES ARE MEAN SD FOR THREE REPLICATES, SIGNIFICANTLY DIFFERENT VALUES
BETWEEN TREATMENTS WITH AND WITHOUT DTT ARE INDICATED (t-TEST; ** FOR e <
.0t~ * FOR P < .05)

Precipitated
protein: protein Precipitated
tannin without protein with
Tannin mass ratio DTT (%) DTT (%)

Carpophyllum 12.5 : 23.6 +_ 2.2 44.2 + 0.6**

Lobophora < 100,000 amu 12.5: 65.6 _+ 5.3 77.2 + 0.7*
Ecklonia 5: 40.1 ___4.1 35.7 + 2.1
Lobophora > 100,000 amu 12.5 : 79.3 +_ 1.7 79.5 _+ 1.7
Sorghum 125 : 62.9 + 5.4 69.0 + 2.2
quebracho 5: 66.9 _+ 2.9 71.7 + t.4
tannic acid 12.5: 92.8 + 3.8 86.3 + 0.4

solutions (pH 4-6). The basic proteins trypsin (pI = 10.5) and lysozyme (pI =
11. I) were precipitated in slightly acidic to basic solutions (pH 4-10).
The nature of the interaction between phlorotannin and protein was further
probed by adding various organic modifiers to the reaction mixture. If tannin-
protein precipitates are stabilized by hydrogen bonds between the tannin and the
protein, then hydrogen bonding solvents should interfere with precipitation. If
hydrophobic forces stabilize the tannin-protein complex, then nonpolar solvents
or detergents should interfere with precipitation. W e found that several different
organic solvents influenced the precipitation of phlorotannins by bovine serum
albumin. The magnitude of the effect was dependent on the properties o f the
solvent and on the phlorotannin (Table 2). In general, the most effective inhib-
itors of precipitation were dimethylformamide and dioxane. The ability of di-
methylformamide to inhibit precipitation of phlorotannin by protein was con-
sistent with its ability to dissolve phlorotannin-protein complexes. Methanol
inhibited the precipitation of only a single phlorotannin, from Ecklonia, and
stimulated precipitation of the high molecular weight phlorotannin from Lobo-
phora.
The effects of the organic solvents on precipitation were concentration
dependent. For example, 10% dimethylfovmamide or dioxane had little effect
 TABLE 2. PRECIPITATIONOF PHLOROTANNINS IN THE PRESENCE OF ORGANIC MODIFIERS. THE INDICATED ORGANIC SOLVENT WAS
SUBSTITUTED FOR PART OF THE BUFFER TO ACHIEVE t 0% SOLVENT IN THE |NCUBATION MIXTURE. ALL INCUBATIONS ALSO CONTAINED
3.7% METHANOL INTRODUCED WITH THE PHLOROTANNIN. VALUES ARE THE MEAN ~ SD FOR THREE DETERMINATIONS. THE DATA ARE
NORMALIZED BY EXPRESSING AMOUNT OF PHLOROTANNIN PRECIPITATED AS A % OF THE AMOUNT PRECIPITATED IN THE ABSENCE OF
ADDED ORGANIC SOLVENT. IN THE ABSENCE OF SOLVENT, 48. 1% OF THE ADDED Ecklonia PHLOROTANNIN WAS PRECIPITATED; 78.4%
OF THE ADDED Carpophyllum TANNIN WAS PRECIPITATED; 5 1.6% OF THE ADDED LOW MOLECULAR WEIGHT Lobophora TANNIN WAS
PRECIPITATED; AND 59.6% OF THE HIGH MOLECULAR WEIGHT Lobophora TANNIN WAS PRECIPITATED

~bophora Lobophora
Solvent Mode of action Ecklonia Carpophyllum 5-100 > IO0

none 100 100 100 100

DMF hydrogen bonding 3.7 +_ 1.3 67.6 2.5 75.7 5:4.9 89.7 1.4
acetone hydrogen bonding 44.1 2.7 87.5:5:3.5 78.6 4.9 105.4 2.2
ethanol nonpolar solvent 39~5 2~2 69.2 + 4~5 66~5 6.2 121.0'~
methanol nonpolar solvent 79.6 6.7 96.8 5.8 98~7 4.7 126.9 6.3
dioxane nonpolar solvent 23.6 _+ 2.0 36.7 4.6 85.3 1.6 90.5 6.0

at/ ~ 2

,,.q

z
PHLOROTANNIN-PROTEIN INTERACTIONS 1889

100
"o [] with D T T Lysozyme & Ecklonia
Q~

80 A without D T T
Q. /
i
tO
/"

~ 60
c /
.. \ x
c-- ', x
~ 40
/i:

o
t-
o. 2O
/ '\

0 ] .......... ~ T I ........ i

0 2 4 6 8 10 12 14
pH
FIG. 4. Precipitation of Ecklonia phlorotannin by lysozyme as a function of pH and
presence of DTT. Tannin and protein were incubated in 0.1 M phosphate buffers without
DTT or in the same buffers containing 15 rnM DTT. Points represent the means of three
determinations, and the error bars represent standard deviations.

on precipitation of high molecular weight l~)bophora phlorotannin (Table 2).

However, if the mixture of phlorotannin and protein contained 20% of either
of these solvents, precipitation of high molecular weight Lobophora phlorotan-
nin was inhibited 60% (dioxane) or 70% (dimethylformamide).
Adding as little as 1 to 3% of the nonionic detergent Triton X t00 to
incubation mixtures containing bovine serum albumin and Ecklonia or Carpo-
phytlum phlorotannin completely prevented precipitation.

DISCUSSION

The effects of tannins or phlorotannins on herbivores varies significantly in

both terrestrial (Bernays et al., 1989) and marine (Steinberg and van Altena,
1992; Steinberg et al., 1995) systems. Some of the basis for this variation is
documented for terrestrial herbivores, and includes characteristics of both the
herbivores and of the tannins (Martin and Martin, 1984; Bernays et al., 1989;
Clausen et al., 1990; Hagen-nan and Robbins, 1993).
In marine systems, the effects of phlorotannins on herbivores vary as a
function of the type of phlorotannin (Steinberg and van Altena, 1992; Boettcher
t890 STERN ET AL.

100 - 100
Pepsin m BSA
m 80~ Carpophytlum N 80 ', i Carpophyllum

i
& Ecklonia
~.60 / Ecklonia
.c c
E
m 40. ~ 4o
&
~ 20, ~ 2o /
A

0 ~ 0
0 2 4 6 8 10 12 0 2 6 8 10 12
pH pH

.~ 100 - 100 i
c d Trypsin
RuBPC
80. i Carpophyllum
~ 80 i Carpophyllum ,
G
e~ I
60~ ~. 60
.c
40, ~ 40

~ 20. ~ 2o
i
0, 0
0 2 4 6 8 10 12 2 4 6 8 t0 12
pH pH

F~G. 5. Precipitation of Carpophyllum or Eckhmia phlorotannin by proteins as a function

of pH. Tannin and protein were incubated in O. 1 M phosphate buffers containing 15 mM
DTT, Points represent the means of three detenninations, and the error bars represent
the standard deviations, a, Pepsin. b, Bovine serum albumin, c, RuBPC. d, Trypsin.

and Targett, 1993), the characteristics of the gut environment (Tugwell and
Branch, 1992), the herbivore species (Steinberg and van Altena, 1992) and the
geography (Van Alstyne and Paul, 1990; Steinberg and van Altena, 1992; Stein-
berg et at., 1995). For the most part, the specific mechanisms of action of
phlorotannins which underlie this variation are unknown.

Reactions of Oxidized Phloromnnins

Appel (1993) proposed that phenolic oxidation may have significant eco-
logical implication and we have obtained detailed experimental evidence for
reactions between oxidized tannins and protein. A mechanism for t~rmation of
covalent complexes between protein and oxidized plant phenols was proposed
by Pierpomt (1969a), and reactions between oxidized simple phenolics, such as
chlorogenic acid, and protein have been demonstrated (Pierpoint, 1969b; Felton
et al., 1992). Leatham et al, (1980) demonstrated that tannic acid treated with
peroxidase and hydrogen peroxide precipitated more protein than untreated tannic
acid. However, evidence accumulated under a wide range of conditions indicates
PHLOROTANNIN-PROTEIN INTERACTIONS 1891

that in the absence of added oxidants, oxidation is not involved in the reactions
between terrestrial tannins and proteins (Haslam, 1989; Hagen-nan, 1992). In
contrast, we have found that the phlorotannins from marine algae spontaneously
oxidize and react with protein to form dark colored complexes resistant to dis-
solution unless a reducing agent is present.
We initially recognized oxidation in the phlorotannin system because of
difficulties we encountered when attempting to redissolve some phlorotannin-
protein precipitates for analysis. Dark-colored precipitates resistant to solubili-
zation were obtained especially when Carpophyllum phlorotannins were incu-
bated with protein in alkaline solution. The precipitates could not be redissolved
by hydrogen bond-breaking solvents, such as dimethylformamide or by protein
denaturants, such as sodium dodecyl sulfate, leading us to postulate that covalent
bonds were stabilizing these complexes. The color of the precipitates was con-
sistent with the orange-brown colors of oxidized phenolics (Pierpoint, 1969a).
We used mild reducing agents to demonstrate the importance of oxidation
in phlorotannin-protein interactions. We found that when 15 mM DTT was
included in the mixture of phlorotannins and protein, light colored precipitates
formed. These precipitates were easily dissolved in dimethylformamide, sug-
gesting that the protein-phlorotannin complex formed under these conditions
involved noncovalent interactions. The reducing agents were effective with a
variety of proteins and phlorotannins over a broad pH range. We obtained similar
results using an alternative reducing agent, fl-mercaptoethanol, in place of DTT.
The method of analysis that we employed (Stem et al., 1996) is particularly
well suited to detecting insoluble covalent complexes between oxidized tannins
and protein. Our method is dependent on resolubilizing the precipitate; irrever-
sible, covalent complexes were readily recognized. Tugwell and Branch (1992)
examined phlorotannin-protein interactions but did not report any effects that
can be attributed to oxidation. They analyzed the supematant after removal of
the precipitate and thus did not assess solubility or other characteristics of the
precipitates. Our study was not designed to examine soluble tannin-protein com-
plexes stabilized by either noncovalent or covalent bonds (Hagerman and Rob-
bins, 1987).
In most of our experiments, more phlorotannins were detected in precipi-
tates formed under reducing conditions than in precipitates formed under oxi-
dizing conditions (Figure 2). This increased precipitation could be an artifact
resulting from the higher color yield of DMBA with reduced phlorotannins. An
additional source of artifact could be the improved solubility of complexes formed
under reducing conditions, since the assay only detects the phlorotannins that
can be redissolved after precipitation. To eliminate these possible interferences,
we performed precipitation reactions using a radiolabeled protein. Precipitated,
labeled protein is determined by direct radiochemical counting so interferences
in the assay are eliminated. For Carpophyllum phlorotannins and moderate
1892 STERn ET AL.

molecular weight Lobophora phlorotannins more protein was precipitated under

reducing conditions than under oxidizing conditions (Table 1). This clearly
demonstrates that the effects of reducing agents on phlorotannin precipitation
are not artifacts of our method of analysis.
Oxidation is more important for interactions of phlorotannins with some
proteins and less important for other proteins. The proteins examined differ in
many respects, including size, amino acid composition and isoelectric point.
The role of oxidation for precipitation of phlorotannins by RuBPC was partic-
ularly variable. RuBPC precipitation of Carpophyllum phlorotannins was
enhanced more by the addition of DTT than was the precipitation of any other
protein, but addition of DTT had no effect on precipitation of high molecular
weight Lobophora phlorotannins by RuBPC. RuBPC is unusually large (560,000
amu) and is a 16-mer comprised of two types of subunits. The other proteins
used in these experiments range in size from 14,400 amu (lysozyme) to 65,000
amu (bovine serum albumin) and are all single polypeptide chains. RuBPC
contains an unusual number of reduced thiols (96 cysteines per 16-mer of spinach
RuBPC), which may make it particularly susceptible to oxidation. Of the other
proteins examined, bovine serum albumin contains 17 disulfide bonds (cystines)
and one cysteine residue: the other proteins contain three to six cystines and no
cysteine.

Tannin Structure and Oxidation Reactions

The tendency of phenolics to oxidize is affected by the nature of substituents
and the position of substituents on the phenolic (Ronlan, 1978). The presence
of an electron withdrawing group such as a carboxylic acid or an ester makes
the phenolic more difficult to oxidize: thus, the redox potential ofpara-hydroxy
benzoic acid is higher than the redox potential of phenol (Table 3). Electron
donating groups such as alkyl, aryl. ether, or hydroxyl groups make the phenolic
easier to oxidize. Among the electron donating groups, alkyl and aryl groups
have the smallest effect, ether groups have a larger effect and hydroxyl groups
have the largest effect on oxidation potential (compare ortho-methyl phenol,
ortho-methoxyphenol and catechol, Table 3). Substituents ortho or para to a
phenolic group change the redox potential more than meta substituents. For
example, the redox potentials of catechol and hydroquinone (ortho and para
substituted, respectively) are much lower than the redox potential of phenol
while the redox potential of resorcinol (recta substituted) is intermediate
(Table 3).
We found that the susceptibility of tannins to oxidation can be explained
by examining the structural characteristics of the tannins. The susceptibility of
some phlorotannins to oxidation is a consequence of extensive substitution by
aryl ethers (1,2, and 3, Figure 1). The ether groups are strong electron donating
PHLOROTANNIN-PROTEIN INTERACTIONS 1893

TABLE 3. REDOX P(Yl'ENTIALS(;I- SIMPLE PHENOIJCS

Redox
Subslitulenl pallem and pt~tenlial
Slrtlclure type (V)

I OH phenol O. 62"
unsubstituled 0 , b 3 ~'

HO
.OH
0
para-hydroxybcnzoic acid
carboxylic acid
electron w ilhdrawing
0.72 ~'

~ OH

CH 3
ortho-mcthyl phenol
alkyl
clectnm donating
O.56~*

~ OH
/" I_1
O ~ ' " ~ ~3
ort/m-mclhoxy phenol
ether
ctcclmn donating
0 . 4 0 ~'

@[~ OH

OH
calechol
o,'rho h.~dmxyl
electron donaling
018"

HO
~ OH para-hydroquinone
para hydroxyl
electron donating
O. t8"

OH resoreinol
meta hydroxyl O.39"
electron donating
OH

"Ew," vs. standard calomel electrode, pH 7 phosphate (Metres & Zuman 1974)
~'EI,, vs. standard calomel electrode, pH 5.6 isopropam~l/H20 (Suatoni ct al. 1961)
1894 STERN ET AL.

groups, especially when they are positioned ortho or meta to phenolic groups
as in the phlorotannins. Ether substituents facilitate oxidation of the resorcinol-
like phenolic subunits of the phlorotannins.
The phlorotannins from Carpophyllum were more susceptible to oxidation
than any other tannin we examined. Both the amount of protein and the amount
of phlorotannins precipitated were enhanced by the addition of reducing agent
to the protein-Carpophyllum mixtures (Figure 3, Table 1). The mixture of phlo-
rotannins found in Carpophyllum includes fuhalols (1. Figure 1) (Ragan and
Glombitza, 1986) and hydroxyphlorethols (2, Figure I) (Glombitza and Li,
1991a). Both of these types of phlorotannin characteristically have ortho hydroxy
groups on the terminal unit. The addition of the strong electron donating hydroxyl
group ortho to the other hydroxyl groups on the terminal unit of the Carpo-
phyllum phlorotannins enhances sensitivity to oxidation. In contrast, the phlo-
rotannins from Ecklonia are less susceptible to oxidation (Figure 2, Table 1);
phlorotannins tom Ecklonia such as phlorethols (3, Figure 1) do not contain
any hydroxyl groups in the orrho orientation (Ragan and Glombitza, 1986). We
found that the phlorotannins from Lobophora, like those from Ecklonia, have a
limited tendency to oxidize, but nothing is known about the structural chemistry
of the phlorotannins from Lobophora.
These results suggest that structural differences between the phlorotannins
may substantially influence the interaction between phlorotannins and proteins.
Consequently, the ability of herbivores to counteract the effects of different
phlorotannins by controlling the redox potential of the gut will also vary. Based
on the limited structural data available, we predict that ether-linked phlorotan-
nins which have ortho substituted hydroxyl grops on one or more rings will
generally be the most susceptible to oxidation. We predict that phlorotannins
which are aryl-aryl linked, such as the fucols (4, Figure 1), will be least sus-
ceptible to oxidation, since aryl groups are weaker electron donating groups than
aryl ethers. This prediction could be tested by using methods like those devel-
oped by Glombitza (e.g., Glornbitza and Li, 1991a; Glombitza and Li, 1991b:
Li and Glombitza, 1991) to determine the structures of isolated phlorotannins,
and then determining the redox potentials of those purified compounds. Our
studies were done with mixtures of phlorotannins, making it impossible to spe-
cifically evaluate the contribution of each type of phlorotannin to oxidation
reactions.
The terrestrial tannins did not appear to oxidize spontaneously (Table 2)
despite the catechol-like arrangement of hydroxyl groups common to these tan-
nins (5 and 6, Figure 1). Examination of the other substituents on the terrestrial
tannins explains their insensitivity to oxidation. For the gallotannins (5, Figure
1), each of the phenolic rings bears at least one electron withdrawing ester group,
diminishing susceptibility to oxidation. The condensed tannins have alkyl sub-
stituents on both the phenolic rings (A ring and B ring of 6, Figure 1). Alkyl
substituents arc poor electron donating groups, and apparently do not enhance
PHLOROTANNIN-PROTEIN INTERACTIONS 1895

oxidizability of the phenolic tings sufficiently to promote air oxidation of the

condensed tannins. It would be useful to directly determine the redox potentials
of various phlorotannins and terrestrial tannins in order to better understand their
relative susceptibility to oxidation,

Noneovalent Phlorotannin-Protein Interactions

Insight into the types of bonds that stabilize tannin-protein complexes can
be obtained by varying the composition of the solution in which tannin-protein
complexes are formed. For example, covalent bonds between oxidized phenolics
and protein are likely to form at high pH since phenolics are more readily
oxidized at high pH (Ronlan, I978). Our observations on covalent complexes
were consistent with that general trend. In the absence of reducing agent, we
detected brown complexes resistant to dissolution at pH values above 4 but not
at more acidic pH values.
The pH profiles we obtained for precipitation of reduced phlorotannins by
proteins are similar to the profiles for precipitation of terrestrial tannins (Hag-
erman and Butler, 1978; Martin and Martin, 1983; Hagerman and Klucher,
1986) or phlorotannins (TugwelI and Branch, 1992) for the same proteins. Pre-
cipitation of proteins generally occurs most readily at pH values near the pl,
because ionic repulsion is minimized at that pH (Mahler and Cordes, 1971).
The general pattern of precipitation by phlorotannins was consistent with that
trend.
High pH should favor either ionic or covalent interaction; at pH values
above about 9 phenolic groups are ionized. Reduced phlorotannins were not
precipitated by most proteins at high pH values. However, trypsin and lysozyme
precipitate some phlorotannin at pH 10 (Figure 3, Figure 4), suggesting either
ionic interactions or oxidation of the phlorotannin even in the presence of DTT.
Further studies are necessary to distinguish between these two possibilities.
In the presence of the reducing agent, phlorotannin-protein interactions
were inhibited by a variety of solvents (Table 2). Inhibition by hydrogen-bond-
ing solvents such as dimethylformamide or acetone provides evidence that
hydrogen bonding plays an important role in precipitation. Dimethylformamide
is a more effective inhibitor because it is a stronger hydrogen bond acceptor
than is acetone.
Hydropbobic forces appear to play a minor role in formation of phlorotan-
nin-protein precipitates. The temperature independence of precipitation (Figure
1) suggests that hydrophobic forces are not important in formation of phloro-
tannin-protein complexes (Kennedy, 1990). Furthermore, the dependence of
precipitation on pH suggests that hydrophobic forces do not stabilize these com-
plexes, since hydrophobic interactions are generally pH-independent. Nonethe-
less, precipitation is inhibited by some nonpolar solvents such as dioxane, and
ethanol inhibited precipitation more effectively than its more polar analogue,
1896 STERN ET AL.

methanol (Table 2). Tugwell and Branch (1992) found that gut surfactants inhib-
ited the interaction between the Fucus phlorotannin and RuBPC and we found
that the nonionic detergent Triton X 100 inhibited precipitation. The effects of
these nonpolar solution modifiers indicates that the interaction between reduced
phlorotannins and proteins involves nonpolar interactions as well as strong
hydrogen bonds.

CONCLUSIONS

This is the first study to compare the reactions of phlorotannins and ter-
restrial tannins with proteins. The work demonstrated that oxidized tannins can
form covalent complexes with proteins, and confirmed the hypothesis that redox
conditions may be an important determinant of the biological activity and fate
of tannins (Appel, 1993). Our study suggests that oxidative reactions are com-
mon among certain phlorotannins, but that oxidation of terrestrial tannins may
be rare. It is logical to conclude that gut redox conditions should be examined
in marine herbivores which consume phlorotannin-containing brown algae. For
example, the redox potential of the gut might be relatively unimportant ['or an
herbivore which consumes Ecklonia, but gut redox potential could be an impor-
tant determinant of the fate and effects of Catpophyllum phlorotannin.
In addition to redox conditions, we found that pH and solution composition
influenced phlorotannin-protein interactions. The phlorotannin-protein com-
plexes which formed in a reducing environment were similar to terrestrial tannin-
protein complexes that have been characterized earlier. Thus, we believe that
in marine herbivores, as in terrestrial herbivores, variation in sensitivity to di-
etary tannins is due to variation in herbivore gut chemistry such as pH, surfactant
concentrations, and presence of tannin-binding proteins (Bernays et al., 1989;
McArthur et al., 1991). We have recently confirmed that concentrations of metal
ions (sodium, potassium, calcium, and magnesium) significantly influence pre-
cipitation of phlorotannins by proteins (Tugwell and Branch, 1992; Mason and
Hagerman unpublished data). These results suggest that marine herbivores could
control the effects of tannins by controlling salt concentration in the gut. We
are currently extending those studies to explore the basis for the metal ion effects.
We have demonstrated that one of the activities of phlorotannins, ability
to precipitate proteins, is related to the chemical structures of the tannins. Similar
structure-activity relationships have been found for terrestrial tannins. Under-
standing how structure influences biological activity is an essential component
to understanding the effects of phlorotannins on herbivores.

Acknowledgments--NancyM. Targett (Universityof Delaware. Lewes. Delaware, 1995)gen-

erously provided purified phlorotannins from l~Jbophora. We appreciated the assistance of Matt
Riebel and Brice Nelson.
PHLOROTANNIN-PROTEIN INTERACTIONS | 897

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