EFFECT OF CONDENSED TANNINS PREPARED FROM SEVERAL FORAGES ON THE IN-VITRO PRECIPITATION OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE (RUBISCO) PROTEIN AND ITS DIGESTION BY TRYPSIN (EC-2.4.21.4) ND CHYMOTRYPSIN (EC-2.4.21.1)

A series of in vitro a experiments was undertaken to determine the ext ent to which Sephadex LH-20 treated extracts from a range of temperate foraged precipitated ribulose-1,5-bisphosphate carboxylase (Rubisco) and affected the enzymatic hydrolysis of Rubisco protein by trypsin an d chymotrypsin at a range of pH values. Rubisco was chosen because it represents the principal dietary protein for ruminants fed fresh forag es. Condensed tannins (CT) or proanthocyanidins (PA) are routinely pur ified by chromatography using Sephadex LH-20 as a matrix. However, the se extracts contained non-CT phenolics together with PG so the term 'C T extract' was preferred to 'PA' to describe the extracts. The in vitr o precipitation of Rubisco provided a means to compare the reactivity of the CT extracts. The amount of CT extract required to precipitate a ll the Rubisco in 10 mu g of total soluble leaf protein from white clo ver (Trifolium repens) when this protein was incubated with CT extract s of Lotus corniculatus, L pedunculatus and sainfoin (Onobrychis vicii folia) was similar, with between 25 and 50 mu g of extract required. T he CT extract of sulla (Hedysarum coronarium) also precipitated all th e Rubisco, however this only occurred with 50 mu g of the extract. The CT extract of dock (Rumex obtusifolius) precipitated all the Rubisco when 5 mu g of extract or greater was incubated with total soluble lea f protein. However, the differences between the reactivity of ah these CT extracts at a range of pH values appeared to be small. Condensed t annin extracts of L corniculatus and L pedunculatus partially inhibite d the hydrolysis of Rubisco by trypsin;and chymotrypsin to a similar e xtent, but the extent of the inhibition was affected by pH. The inhibi tion was greater at pH 6.0 than 7.0, whilst at pH 8.0, CT extracts had little or no affect on trypsin and chymotrypsin. It was concluded tha t, although the precipitation of Rubisco provided an ideal method for comparing CT extracts, reactivity alone was unlikely to account for th e differences in nutritive value that occur with forages containing CT . (C) 1998 SCI.