323

CHEMISTRY OF TANNIN-PROTEIN COMPLEXATION

Ann E. Hagerman

Department of Chemistry
Miami University
Oxford, Ohio 45056

ABSTRACT

The importance of tannin-protein interactions in leathermaking and in

plant-animal interactions has long been recognized, but detailed knowl-
edge of the chemistry of the interaction has only recently become avail-
able. Tannin-protein interactions are influenced by characteristics of the
protein (including size, amino acid composition, pI, and extent of post
translational modification), characteristics of the tannin (size, structure,
heterogeneity of the preparation), and conditions of the reaction (pH,
temperature, solvent composition, time). The most familiar tannin-
protein interactions result in precipitation of the complex, but soluble
complexes also form under certain conditions. Both soluble and insol-
uble complexes are stabilized by reversible, non covalent bonds between
tannin and protein.

INTRODUCTION
The role of tannin in diverse processes, including ecological interactions, manu-
facturing, and food processing, is at least in part a consequence of the tendency of
tannins "to precipitate alkaloids, gelatin and other proteins".l Historically, leather
manufacture depended upon treatment of animal skins with tannin-containing plant
preparations to form insoluble, microbe-resistant products. Interactions of colla-
gen and gelatin with plant tannins during leather making have been investigated,2
as have interactions of proteins with phenolics during beverage preparation from
fruits and grains. 3 The suggestion that tannins protect plants from herbivores
by complexing with protein and thus reducing the digestibility of the tannin-
containing plants 4 ,5 has influenced ecologists for a decade and has stimulated many
investigations of tannin-protein interactions. 6 Similar ideas have been pursued by
nutritionists and agronomists interested in the effects of tannin on humans and

R. W. Hemingway et al. (eds.), Chemistry and Significance of Condensed Tannins

© Plenum Press, New York 1989
324 Hagerman

domestic animals. 7 The efforts of plant biochemists to overcome enzyme inhibition

by phenolics in plant extracts have yielded substantial insight into tannin-protein
interactions. S,9
For the earliest investigations of tannin-protein interactions, pure tannins and
proteins of defined structure were unavailable, but those problems have been re-
solved by newer methods of purification and structure determination. No attempt
has been made in this review to summarize the older literature. Instead, only
investigations of the interactions of condensed tannins (proanthocyanidins) with
proteins have been included. Detailed characterization of the interactions between
hydrolyzable tannins and protein can be found in recent publications from Haslam's
laboratory.10,1l The formation of insoluble proanthocyanidin-protein complexes will
be the primary topic of this review, since descriptions of insoluble complexes dom-
inate the literature.
NATURE OF THE TANNIN-PROTEIN INTERACTION
Tannin-protein complexes are normally established by hydrogen bonds and hy-
drophobic interactions, without contribution from covalent or ionic bonds. It has
long been believed that hydrogen bonds are involved in formation of tannin-protein
complexes,S,12,13 and the importance of those bonds is demonstrated by the ability
of hydrogen-bonding solvents such as formamide and its N-substituted derivatives
to disrupt the tannin-protein interaction. 14 The pH dependence of protein precipi-
tation by condensed tannin suggests that the phenolic hydroxyl acts as a hydrogen
bond donor, since at pH values above the pKa of the phenolic hydroxyl group
( ...... pH 10), proteins are not precipitated by condensed tannin. 15 ,16 The carbonyl
oxygen of the peptide bond presumably serves as the hydrogen bond acceptor.
Hydrogen bonding is augmented by hydrophobic interactions between nonpolar
domains of certain proteins and the nonpolar regions of the tannin molecule,14,17
particularly at acidic pH .10 The only ionizable group in condensed tannins is the
phenolic group, but tannin-protein interactions at pH values above the pKa of that
moiety have not been documented,15,16 leading to the conclusion that ionic inter-
actions between tannin and protein are not important. Under oxidizing conditions,
phenolics react covalently with nucleophilic amino acid side chains such as lysine
or cysteine. 1S However, tannin-protein complexes can be dissociated with a variety
of mild treatments that do not disrupt covalent bonds, including treatment with
detergents, phenol, some organic solvents, or with excess caffeine. 19 - 23 The ease
of disruption of the complexes suggests that covalent bonds do not normally form
between condensed tannin and protein.
The complexes that form between tannin and protein can be either soluble or
insoluble. Tannin and protein are both multivalent binding agents, with many
sites for hydrogen bond formation or hydrophobic interaction. At optimum ratios
of tannin to protein and at optimum pH values, the cross-linked complexes that
form are macromolecular and insoluble in aqueous solution. 10 ,24 The optimum pH
for formation of insoluble complexes is usually around the isoelectric pH of the
protein. 15 At that pH, electrostatic replusions that prevent aggregation of protein
are minimized.