Narcissus and Daffodil

382 E.J.M. Van Damme and W.J. Peumans
PURIFICATION OF NARCISSUS LECTINS
Since preliminary experiments with crude extracts from Narcissus indicated that
they contained an agglutinating factor that can be inhibited only by mannose, a
purification scheme based on affinity chromatography on immobilised mannose
was developed to purify the lectin. Although the affinity purified lectin was virtually
pure, as could be judged from sodium dodecyl sulphate – polyacryl amide gel
electrophoresis (SDS-PAGE), additional hydrophobic interactions chromatography
and ion exchange chromatography were included to ensure complete purity of
the lectin (Van Damme et al., 1988). The same protocol was used to purify the
lectin from different species and cultivars of Narcissus. Furthermore, the protocol
was also applicable for the purification of the lectin from different tissues of
narcissus, e.g., bulbs, leaves and ovaries.
CHARACTERISATION OF NARCISSUS LECTINS
Molecular structure
The molecular structure of narcissus lectin was determined using SDS-PAGE, gel
filtration and ultracentrifugation. Upon SDS-PAGE the lectins isolated from all
Narcissus species and cultivars yielded a single polypeptide band of 12.5 kDa. Since
the results were identical when electrophoresis was conducted either in the presence
or absence of β-mercaptoethanol, it can be concluded that the lectin subunits
are not held together by disulphide bonds (Van Damme et al., 1988).
Gel filtration experiments on a Superose 12 column using a phosphate buffer
containing 0.2 M mannose (to avoid binding of the lectin to the matrix) demonstrated
that the lectins isolated from narcissus elute with an apparent molecular
mass of 25 kDa, indicating that they are probably dimers. Because aspecific interactions
of lectins with the gel filtration matrix often occur and cannot be abolished
completely by the addition of the specific sugar to the running buffer, the molecular
mass was also determined by ultracentrifugation of the lectins. Since the
molecular mass of the lectin was calculated to be 36 kDa after centrifugation, the
lectin may be a trimer (Van Damme et al., 1995).
It should be indicated here that all lectins isolated from different species and
cultivars of Narcissus show the same molecular structure and biochemical characteristics
(E. Van Damme, unpublished data).
Detailed analyses of the narcissus lectins have shown that the amino acid
composition of these lectins is typified by high contents of asparagine/aspartic acid,
threonine, glycine, serine, glutamine/glutamic acid and leucine. The lectins
contained no amino sugar and only low levels of neutral sugars, most probably
contaminants, indicating that the lectins are not glycosylated (Van Damme et al.,
1988, 1991a).
Narcissus lectins are complex mixtures of isolectins
When the purified lectins from different Narcissus species were analysed by high
resolution ion exchange chromatography it became evident that they all yielded a