of Bougainvillea. Roots of Bouginvillea were therefore
extracted as described in the experimental section.
The majority of the antiviral activity was recovered
from a 40-90% ammonium sulphate fraction. The
pellet was redissolved in buffer A, dialysed against
the same buffer and the dialysate lyophilised. The
lyophilized powder was dissolved in buffer B and was
fractionated by CM-Sepharose cation exchange chromatography
(Fig. 1A) where fractions 51-54 eluting
at about 150 mM NaC1 contained antiviral activity.
The pooled fractions were lyophilized, dissolved in
buffer C and further fractionated by DEAESepharose
anion exchange chromatography, where
antiviral activity came through in the unbound fraction.
The fraction was pooled, adjusted to pH 6.0 and
was resubmitted to CM-Sepharose cation exchange
chromatography (Fig. 1 B) where antiviral activity was
detected in fractions 44-48 which also coincided with
the point where bound protein eluted. RP-HPLC of
active fractions from the Fig. l A pool (Fig. 1 C; Trace
A) and from the Fig. 1B pool (Fig. 1C; Trace B)
separated into two protein peaks, respectively.
However, enzyme assay of fractions from the two
HPLC separations showed that only the first elution
peaks had the same retention times and had BAP I
antiviral activity. This purified material was used to
characterise the active protein.