Analysis of a ferric leghemoglobin reductase from cowpea (Vigna ...

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though approximately 50% of the total activity
was lost. A 40-fold decrease in the ratio of diaphorase
to FLbR activity (Table 1) indicated that
many of the other contaminating diaphorases were
removed at this step. On the FPLC anion exchange
column, FLbR was eluted as a sharp peak
at about 15% of NaCl gradient (150 mM NaCl,
Fig. 1). The specific activity of the purified cowpea
FLbR was about 50% of that reported for the
soybean enzyme [7]. The difference in the specific
activities between the cowpea and soybean FLbRs
could result from inherent differences in the two
enzymes.
P. Luan et al. / Plant Science 154 (2000) 161–170
Table 1
Purification and specific activities of FLbR from cowpea nodule cytosol a
3.2. Homogeneity and molecular weight analysis
of cowpea FLbR
Samples collected from each purification step
were subjected to 10% SDS-PAGE and silver
staining (Fig. 2). The sample after the Sephacryl
S-200 step (lane 4) exhibited a single distinct
protein band of about 55 kDa. The molecular
weight for native cowpea FLbR was estimated to
be 110 kDa using the Sephacryl S-200 SF column
(data not shown) and thus appears to be a homodimer.
These molecular weights were similar to
those reported for soybean FLbR [7].
Steps Total protein DCIP reductase (A) specific activity
Ratio A/Bd FLbR (B) specific activity Total FLbR
(mg) (U mg−1 ) c
(U mg−1 ) b activity (U)
Crude extract 893 187 0.22 194
850
G-25 607 254 0.29 176 875
Hydroxylapatite
4.5 368
17.3
78
21.3
Mono-Q 0.31 2289 162 50 14.1
S-200 0.15 2565
216 32 11.9
a Purification steps are described in Section 2.
b One unit is defined as 1 nmol of DCPIP reduced per min.
c One unit is defined as 1 nmol of Lb 2+ O2 formed per min.
d This is the ratio of DCPIP reductase (diaphorase) specific activity to FLbR specific activity.
Fig. 1. Separation of FLbR by an ion-exchange FPLC on a Mono-Q HR5/5 column. The column was equilibrated with Buffer
A (50 mM Tris–HCl, pH 7.5), and eluted with a linear NaCl gradient from 0 to 35% of Buffer B (1 M NaCl, 50 mM Tris–HCl,
pH 7.5) in 50 ml total volume (Buffer A plus Buffer B) at a flow rate of 1 ml min −1 . Cowpea FLbR was eluted at about 15%
of the gradient (150 mM NaCl, as indicated by the arrow).