lution (0 to 0.1% w/v, final concentration) were added to 5 ml of a 1 mg m l - 1 protein solution. The mixture was vortexed and allowed to stand for 15 min. Except for the viscosity determinations, the tannin-protein-complexes were precipitated from the solution by centrifugation (12500 g, 15 min, 4 ~ C) and the supernatant (protein-tannin solution) was used for protein determinations.
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freshly extracted plant protein was used. Plant protein was extracted from 150 g two-week old Pisum sativum leaves grown in vermiculite with Long Ashton nutrient media (Hewitt 1952) receiving 200 rag-nitrate N 1-1 in the green house. Leaves were homogenized in a Waring blender with 300ml 100mM phosphate buffer (pH7.5), squeezed through 4 layers of cheese cloth and centrifuged at 10 000 g for 20 min. The supernatant was brought to 60% saturation with (NH4)zSO 4 and the denatured protein isolated by centrifugation (10000 g, 20 min). The protein pellet was redissolved in a minimum of extraction buffer, desalted on a 15 x 1.5 cm Sephadex G25 columns preequilibrated with extraction buffer, and concentrated by freeze drying. All operations were carried out at 4 ~ C. Protein solutions were made up in either a 0.2 M acetate buffer (pH 5.0) or a 0.1 M phosphate buffer (pH 7.0).
Protein determinations. Protein content was determined by
either absorbance at 280 rim, the Folin-Lowry assay (Lowry
421 et al. 1951), biuret assay (Gordon et al. 1977) or by the standard micro-Kjeldahl technique with selenium and copper catalysts.
Protein source.and
Tannins (phenolic compounds, 500 to 3000 daltons) have been implicated in animal nutrition (Moran and Hamilton 1980), plant - herbivore interactions (Milton 1979; McKey et al. 1981), plant resistance to infection (Levin 1976) and fruit or seed maturation (Joslyn and Goldstein 1964). In ecological or nutritional studies it is often advantageous to quantify both the amounts of tannin and protein present in samples. Such investigations have been limited by methodology. Tannin determinations are deficient as these techniques depend on a colour development which is related to the tannin molecular weight, form, structure and concentration (Martin and Martin 1982), while the techniques used to determine protein concentration are problematic in the presence of tannic compounds (Marks et al. 1985). The phenolic hydroxyl groups of tannins form stable cross-links with proteins (Walker 1975) and would denature proteins by altering the three dimensional shape. An alternative method to measure tannin/phenolic content has been developed where the amount of protein precipitated from a solution by the addition of tannins/phenols is taken to be a measure of the amount of tannin/phenols present. Such an assay system would be most appropriate in studies of the significance of tannins in herbivory (Martin and Martin 1982). The relationship between tannin concentration and the amount of protein precipitated in unclear. Jones and Mangan (1977) and Griffiths and Jones (1977) found an
A.M. Amory and C.L. Schubert Biology Department, University of Natal, Durban, 4001, South Africa Summary. Protein determinations on protein-tannin complexes after protein isolation (gel filtration and trichloroacetic acid [TCA] precipitation) or phenolic extraction (polyvinyl pyrrolidone [PVP] and organic solvent precipitation) were unsuccessful. Kjeldahl determinations of the amount of unprecipitated protein bovine serum albumin [BSA] showed a sigmoid relationship with increasing concentrations of tannins. A similar relationship was found for the reduced viscosity of BSA and plant protein, and the concentration of tannin. Non-linear regression and curve normalization allowed three variable (kl, ka and T1/2) to be defined for the quantification of the protein-tannin interaction/s. Such a treatment may be useful in studies of the role of tannins in plant-herbivore interactions. Key words: Tannins - Proteins - Interactions - Quantification exponential relationship, while Martin and Martin (1982) found it to be linear. Little is known of the denaturation of proteins by organic compounds but it can be assumed to be nonlinear (Lepanje 1978). In this study a number of techniques were tested to investigate and quantify the relationship between the amount of tannin present and the amount of protein precipitated by these tannins. Materials and methods