Chemistry Reference
In-Depth Information
Conversely, considering DEAE-dextran, complex formation is stabilized when
the Coulomb forces are large (high affinity between E and S, and the fact that [S] is
small does not have a direct influence on the complex formation). As
K
m
is small,
K
m
thus conversely becomes negligible in comparison to [S]. Assuming that
K
m
[S], the complex concentration similarly becomes:
½
¼½
0
ES
E
(6.43)
This indicates that complex formation is proportional to DEAE-dextran concen-
tration. In other words, it is likely that there is no significant influence on a
quantitative complex formation by DEAE-dextran concentration, even when the
concentration is very low.
However, the Michaelis-Menten complex formation reaction between DDMC
and DNA is thought to be significantly influenced by concentration. The relation-
ship is expressed in Figs.
6.34
and
6.35
using
K
1
10
7
¼
1.055
(
μ
g/well) and
10
5
(
K
2
¼
μ
g/well), respectively, as determined at the maximum RLU
values, and normalizing the RLU values by taking the maximum experimental
values as 100 %. Figures
6.34
and
6.35
show a good correspondence with both
DEAE-dextran and DDMC under the conditions of 48 h and 0.075
μ
g of DNA. For
the concentration represented on the horizontal axes of Figs. 6.34 and 6.35, using
0.075
1.626
μ
g DNA and 0.75
μ
g DDMC, with a total volume of 30
μ
L for the medium
not containing serum, the DNA concentration is 0.075
μ
g/30
μ
L or 0.0025
μ
g/mL.
The DDMC concentration is 0.75
L. Although the vertical
axis in Fig.
6.35
should be normalized to the amount of complex, because of this
proportion in the amount of complex to RLU, the reaction mechanism may be
understood to be analogous if the trend shown in the figure is similar. As shown in
Figs.
6.36
and
6.37
, transfection of COS-7 cells with samples of DDMC support the
Michaelis-Menten complex formation.
Figure
6.36
shows transfection of COS-7 cells with samples of DDMC having a
grafting rate of 130 % and including 0.075, 0.150 and 0.30
μ
g/30
μ
L or 0.025
μ
g/
μ
μ
g of DNA in compari-
son with calculation values for 0.075
g of DNA by Eq. (
6.38
) at 48 h. The
relationship between RLU values and the amount of DDMC with 0.075, 0.150
and 0.30
μ
g of DNA are also in good accordance with the pattern from calculated
values. The degree of transfection of COS-7 cells in Fig.
6.36
are in the order
0.075
μ
g of DNA, depending on its concentration by diffusion
control. The maximum peak of transfection transfer to high concentration of
DDMC is in the order 0.075
>
0.150
>
0.30
μ
g of DNA.
Figure
6.37
shows transfection of COS-7 cells with samples of DDMC having a
grafting rate of 130 % and including 0.15
<
0.150
<
0.30
μ
μ
g of DNA for incubation times of 48, 72
and 96 h.
The relationship between RLU values and the amount of DDMC with incubation
times of 48, 72 and 96 h are also in good accordance with the pattern from
calculated values in Fig.
6.36
.
The degree of transfection of COS-7 cells in Fig.
6.37
are in the order
72
>
96
>
48 h incubation time.
