Biology Reference
In-Depth Information
280 nm
80
1.0
450 nm
0.9
70
366 nm
0.8
ε
450 nm
= 74 mM
-1
cm
-1
60
0.7
50
0.6
ε
366 nm
= 60 mM
-1
cm
-1
40
0.5
0.4
30
0.3
20
556 nm
0.2
10
0.1
0.0
0
250
300
350
400
450
500
550
600
650
700
Wavelength (nm)
Fig. 2
Electronic absorbance spectrum of Fe(III) heme-bound NC1 dimer. The
solid line
, corresponding to the
left y
-axis, shows the relative absorbencies of the
heme and protein peaks. The
dashed line
, corresponding to the
right y
-axis,
shows the extinction coefficients of the heme, as determined using the pyridine
hemochromagen assay as recently reported [
30
]
DGCR8 ligates to the Fe(III) using two Cys352 side chains contributed
by both subunits; this coordination configuration results in charac-
teristic absorption peaks at 366, 450, and 556 nm (Fig.
2
) [
5
].
Recently, we show that Fe(III) heme activates dimeric apoNC1 for
pri-miRNA processing in vitro, whereas Fe(II) heme does not [
6
].
Dimerization and heme binding are likely conserved properties of
DGCR8 in all vertebrates and at least some invertebrates such as the
star fish
Patiria miniata
[
30
]. Heme and the heme-binding domain
appear to be important for pri-miRNA processing both in vitro and
in vivo ([
4
,
6
,
29
] and our unpublished data), though their physio-
logical functions have not been determined.
In order to obtain consistent results in studying pri-miRNA pro-
cessing and to interpret them properly, it is important to express and
purify recombinant DGCR8 protein with optimal heme content.
When NC1 is overexpressed in
E. coli
, a heme-deficient condition is
generated and hence some heme-free protein is produced [
4
]. The
heme-free NC1 may appear as dimer and monomer [
4
,
6
]. At least a
part of the latter species is actually heterodimer of NC1, in which a
subunit is cleaved by bacterial proteases during overexpression and/or
purification so that only a small fragment (the dimerization domain)
is left bound to the intact subunit [
29
]. The heme content of DGCR8
is indicated by the
A
450 nm
/
A
280 nm
ratio, if the protein is purified to be
free of nucleic acids from expression hosts. It is also possible to pre-
pare apoNC1 from Fe(III) heme-bound NC1 via reduction and heme
removal for studying activation of DGCR8 by heme [
6
]. However,
the preparation of apoNC1 is beyond the scope of this chapter.
