An interesting, unpublished observation, which would deserve closer investigation, was made by
Yann Gohon during his Ph.D. work (Gohon 2002): as could be expected, the migration of cytochrome
b 6 f/A3-70 complexes in sucrose gradients was found to depend on the nature of the counterion present
in the buffer (in these experiments, either Li
+ , Na
+
, K
+
, or Cs
+
). A comparison between Li
+ and Cs
+ is
shown in Fig. 9.6. Based on the mass ratio of bound A8-35 to BR determined by AUC and SANS (see
§ 9.3.8.2), it can be estimated that some 150 counterions comigrate with each BR/A8-35 complex, so
that substituting Cs
+ for Li
+ in the buffer increases the dry mass of the complexes by >20%, assuming
the binding of APol not to change. In Fig. 9.6, the complexes used are cytochrome b 6 f/A3-70
complexes, whose MP/APol mass ratio has not been determined but is certainly higher, leading to a
lesser effect of the Li
+
/Cs
+ substitution. Amphipol A3-70 was used because of its higher density of
charges (twice more than A8-35), which is similar to that of A8-75 or SAPols. Qualitatively, this effect
Fig. 9.4 Distribution of BR and [
3
H]A8-35 upon two successive 22-h centrifugations in 12–20% (w/w)
sucrose gradients. Fractions 10–13 from a first gradient (panel A) were pooled, concentrated, and washed
free of excess sucrose by microfiltration before re-loading and centrifuging onto a second, identical
gradient (panel B). Experimental data: “% BR” (—○—), percentage of native BR in each fraction
(on the basis of A 554 ), expressed with respect to the overall amount of BR in the whole gradient (from
A 280 ); “cpm” (—✶—), [
3
H]A8-35 distribution. Breaking up the distribution of [
3
H]A8-35 into free and
bound fractions: inferred contributions of free (À À À À ) and BR-bound (– – –) APol, assuming a
constant ratio of [
3
H]A8-35 to BR in the complexes. Panels A
0 and B
0 : mass ratio of (○) [
3
H]A8-35/native
BR (from A 554 ) and of (△) [
3
H]A8-35/total protein (from A 280 ) (From Gohon et al. 2008, # 2008 The
Biophysical Society. Published by Elsevier Inc. All rights reserved).
9.3 A Walk Through Selected Solution Studies
415
Yann Gohon during his Ph.D. work (Gohon 2002): as could be expected, the migration of cytochrome
b 6 f/A3-70 complexes in sucrose gradients was found to depend on the nature of the counterion present
in the buffer (in these experiments, either Li
+ , Na
+
, K
+
, or Cs
+
). A comparison between Li
+ and Cs
+ is
shown in Fig. 9.6. Based on the mass ratio of bound A8-35 to BR determined by AUC and SANS (see
§ 9.3.8.2), it can be estimated that some 150 counterions comigrate with each BR/A8-35 complex, so
that substituting Cs
+ for Li
+ in the buffer increases the dry mass of the complexes by >20%, assuming
the binding of APol not to change. In Fig. 9.6, the complexes used are cytochrome b 6 f/A3-70
complexes, whose MP/APol mass ratio has not been determined but is certainly higher, leading to a
lesser effect of the Li
+
/Cs
+ substitution. Amphipol A3-70 was used because of its higher density of
charges (twice more than A8-35), which is similar to that of A8-75 or SAPols. Qualitatively, this effect
Fig. 9.4 Distribution of BR and [
3
H]A8-35 upon two successive 22-h centrifugations in 12–20% (w/w)
sucrose gradients. Fractions 10–13 from a first gradient (panel A) were pooled, concentrated, and washed
free of excess sucrose by microfiltration before re-loading and centrifuging onto a second, identical
gradient (panel B). Experimental data: “% BR” (—○—), percentage of native BR in each fraction
(on the basis of A 554 ), expressed with respect to the overall amount of BR in the whole gradient (from
A 280 ); “cpm” (—✶—), [
3
H]A8-35 distribution. Breaking up the distribution of [
3
H]A8-35 into free and
bound fractions: inferred contributions of free (À À À À ) and BR-bound (– – –) APol, assuming a
constant ratio of [
3
H]A8-35 to BR in the complexes. Panels A
0 and B
0 : mass ratio of (○) [
3
H]A8-35/native
BR (from A 554 ) and of (△) [
3
H]A8-35/total protein (from A 280 ) (From Gohon et al. 2008, # 2008 The
Biophysical Society. Published by Elsevier Inc. All rights reserved).
9.3 A Walk Through Selected Solution Studies
415