BIOCHEMICAL
Vol. 85, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
December 29,1978
TITRATION
CURVES OF INTERACTING
HEMOGLOBIN BY ISOELECTRIC PIER GIORGIO RIGHETTI',
ODepartment
GERARD GACON", ELISABETTA and JEAN-CLAUDE KAPLAN"
of Biochemistry,
Via
Celoria
"Institut
de Pathologie
et U.129,
24,
CYTOCHROME b5 AND
FOCUSING-ELECTROPHOREESIS
DANIELE LOSTANLEN"
GIANAZZA',
1575-1581
University
2, Milan0
20133,
Moldculaire,
rue du Faubourg
of Milano, Italy
Groupes
INSERM U.15
Saint-Jacques,
75014 PARIS
France Received
November
14, 1978
SUMMARY: A strong interaction between cytochrome demonstrated by titration curves in isoelectric The pH of maximum interaction is in the pH range predominant role of Lys of met hemoglobin in the acids of cytochrome b5. The stoichiometry of the (cytochrome b5: hemoglobin subunit) with similar and 8 chains.
b5 and hemoglobin has been focusing - electrophoresis. 8.0-8.3, which suggests a binding to acidic amino complex appears to be 1:l binding affinities for a
INTRODUCTION:
idea
By further
(l),
we have recently
rial
representation
resis
parallel
(such possible
gel.
curves
in the mutant
that
revealed
phenotype
as hemoglobins to correlate
(2).
which
curve
and its
pH 3-10
Moreover,
1575
were
run
bed of in
of the respective
had been
any given similar
electropho-
in a flat
mutants
acid
mobility
ABBREVIATIONS: met Hb = met hemoglobin; isoelectric focusing; UV = ultraviolet.
a picto-
the shape
amino
of very
the electrophoretic
to obtain
by performing
genetic
within
or cytochromes)
et al.
preformed
gradients,
charged
of Rosengren
is possible
pH-gradient
When a protein stationary
it
titration
to an Ampholine
in these
pa-mobility
an original
demonstrated of a protein
perpendicular
polyacrylamide
developing
family
size
of proteins
and shape,
at any given
Cyt b5 = cytochrome
substituted
b5;
it
was
pH with IEF =
0006-291X/78/0854-1575$01.00/0 Copyright 0 1978 by Academic Press, Inc. AN rights of reproduction in any form reserved.
BIOCHEMICAL
Vol. 85, No. 4, 1978
AND BIOPHYSICAL
the number
of protons
bound
frictional
resistance
of the proteins
within
a given
technique
family
the bound
their
complexes
species
the
We have
isolation
that
duction
(4).
red-blood keeps
cell,
range
phosphates,
as well
to see
ones,
in fact
able
and to iso-
such as inositol their
half-life,
as the stoichiometry
on the use of this
metHb and soluble did
of
the kinetics
(5),
oxidized
the half
de-
of metHb reis
reduced
that
in the
soluble
Cyt b5 is
b5 reductase
of the splitting
to determine
was recently
interaction
and Passon
of NADH-Cyt
it
species.
in the course
of this
In turns,
technique
protein
Cyt b5 since
interact
significance
any metHb formed.
possible
unliganded
of interacting
two proteins
form
in order
and to measure
data
as shown by Hultquist
by a soluble
was indeed
organic
we report
The biological
By following it
We were
the present
(3).
article
these
reducing
reduced
studies.
the
constant
recently,
of proteins
of the complex
as a model
More
from
and characterization
chosen
monstrated
(2).
since
is essentially
be resolved
with
complex
In the present
the gel
hexasulphate,
of stability
protein-ligand
in
states
properties
and inositol
the pH range
for
could
of hemoglobin
hexaphosphate
the
to liganded
physico-chemical
late
by the macromolecule,
of macromolecules
was extended
whether
or released
RESEARCH COMMUNICATIONS
Cyt b5
continuously
(5). of the Cyt b5-metHb
life
of the complex
complex,
and the pH
of maximum stability.
MATERIALS AND METHODS: MetHb was prepared from pure HbA by ferricyanide oxidation. Homogeneous soluble Cyt b5 (t-b5) was obtained from trypsin-treated rat liver microsomes according to Omura and Takesue (6). The Cyt bg was in fully oxidized state as checked by spectral analysis. The two freshly prepared proteins were desalted and mixed in a 1:l molar ratio (Cyt bg:Hb subunit) just prior to electrophoresis. The two-dimensional IEF-electrophoresis technique (Z), pH measurements (7) and gel staining (8) were performed as previously described. The spectrophotometric studies were carried out on a Beckman Acta III spectrophotometer. RESULTS AND DISCUSSION: and Cyt b5,
respectively,
temperature,
gel
in this
there
case
Figs. run
strength is
1A and C report
the titration
singly
under
and ionic
no perturbation
in a gel strength
conditions.
of the classical
1576
curves
identical It
of metHb
voltage,
time,
can be seen that
sigmoidal
shape
of
BIOCHEMICAL
Vol. 85, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-+
C
9
IEF
+ t
7
5
Figure 1. Titration curves of metHb (A), Cyt b5 (C) and of a mixture of 150 ug each of metHb and Cyt b5 (B). The small arrows in A, B and C indicate the sample application trough. The gel position where the sample crosses the application pocket represents the zero-mobility plane of each macromolecule, i.e. its isoelectric plane. The gel contains 6% acrylamide, 2% Ampholine pH 3.5-10, 0.2% Ampholine pH 6-8, 5 mM Asp, 5 mM Glu and 5 mM Lys. Running conditions: LKB Multiphor 2117 chamber run at 13 W (600 V at equilibrium), with an LKB constant wattage power supply, for 90 min at 4"C, in the first dimension (IEF). Second dimension: 15 min run at 600 V (constant voltage), 4'C. Staining with a colloidal dispersion of Coomassie Brilliant Blue G-250 in 12% CC13COOH, as in ref. 8. The splitting of the upper curve in B (as in C) is due to inherent heterogeneity of the Cyt b5 preparation. The horizontal line between pH 4 and 7 represents part of the mixture of metHb and Cyt b5 precipitated in the application pocket, possibly due to further aggregation and concomitant sedimentation of the complex in this pH range. The bidirectional arrows and + and - symbols represent the direction and polarity of isoelectric focusing (IEF) and electrophoresis (El.). each the
titration Cyt b5 curve
ves as an anion),
curve. is
On the contrary, strongly
while
deviated
when running above its
the same happens
1577
for
the mixture
p1 (i.e.
metHb,
but
(Fig.
lB),
where
Cyt b5 beha-
especially
below
BIOCHEMICAL
Vol. 85, No. 4, 1978
its
pl
(i.e.
where
metHb behaves
as the formation though
within
can enter
the
mum distance of
of a strong the
complex
gel
only
the complex
with amino
acids
between pH range
ved.This tion) the
of Lys residues In fact
be accounted
of a repulsive Cyt b5 molecule.
This
is
between
Cyt b5 and Cyt c. That
strated
by running
(PI
5),
well
No interaction exhibited
known
for
its
was apparent
in
between
dity, is
observed added
creased. this
the
unperturbed
upon mixing
to a solution Since
the
can probably
due to the formation
is
titration
two proteins be ascribed
(4). again
each
Moreover,
1578
invol-
deprotona-
has been demonacidic
protein
of ligands. each protein
has already formation
in Fig.
been of a com-
increase
absorbance
complex.
of
interaction
in turbi-
2, when metHb
at 700 nm is markedly
light
on
shown).
by the
As shown
to an increased
of an intermolecular
(not
the
confirmed
minimal
on the
aspecific
other
obser-
located
in the mixture
the absorbance exhibit
is
the predictions
(10)
curve
the two species.
of Cyt b5,
possibly with
a 6-8
is
a multitude
since
with
over
of interaction
a rather
of binding
spectroscopy
two proteins
is not
case,
constant
interaction
at pH 8 (via
serum albumin,
this
at pH 10 as well
of maximum stability
of Ng et al.
ability
consistent to acidic
of the ionic
residues
the binding
they
and Glu and Asp large-
agreement
metHb and Cyt b5 do interact
shown by W difference plex
data
as
the mini-
is
split
suppression
by His
metHb and bovine
a completely
That
by the
and the experimental
readily
type
in excellent
(9)
Thus,
behavior
be roughly
pH range
generated
Salemme
ea.
should
an additional
for
force
is
the strength
a narrow that
This
deprotonated
However,
that
so that
of metHb in the binding
complex
groups
appears
the pH of maximum stability
8.0-8.3).
be partly
and carboxyl
pH 8 indicates could
the
Lys would
and the fact
ved around
pH range
respectively.
E-amino
of the complex.
phenomenon
It
the other,
represents
role
where
ly protonated,
two curves case,
of Cyt b5.
as at pH 3.5,
disaggregation
this
the two species.
neutralizes
this
a predominant
RESEARCH COMMUNICATIONS
We interpret
between
each protein
the
(in
as a cation).
complex
upon
between
AND BIOPHYSICAL
scattering
in this
in-
region,
phenomenon,
On the one hand,
as jud-
BIOCHEMICAL
Vol. 85, No. 4, 1978
OD
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
7Wnm
I
OA-
0.2 -
I..
10
5
15
.)
20
(HbLro’ M
Figure 2. Turbidimetric study of the metHb-Cyt bg interaction. A Beckmann a 100 uM soActa III spectrophotometer was used, zeroed at 700 nm against lution of Cyt bg. To 200 ul of this Cyt bg solution, as well as to the blank (dist. water), 2 ul of 1 mM metHb were added at each point of the turbidimetric curve, up to the 10th point, after which 4 ul increments were added. No corrections were made for the sample dilution, since the of Cyt bg is negligible. 7oonm absorbance
ged from
the maximum of the turbidimetric
stoichiometry
of 2:l
and above
this
ratio.
the stoichiometry is
(Cyt b5:Hb
On the other
by the fact
(Fig.
presence
1B).
Moreover,
of an excess
the titration
curve
on of a tight
complex
ble
to assume that
titration this
1:l
complex
is
have a
and seems to be redissolved
below
from
when the
to be 1:l curve
two samples
when the titration
be observed.
requires
a 1:l
This
obtained
each Cyt b5 molecule the physiological
again
stoichiometry.
complex
one is
1579
titration
binds highly
experiments,
(Cyt b5:Hb of free are
subunit).
Thus,
in a 1:l
were
no clear implies
This
Cyt b5 and free
loaded
experiments
of one of the two proteins, could
the complex
the
no titration
in the stable
experiments,
hand,
would
appears
that
metHb can be seen in the gel ratio
subunit)
of the complex
demonstrated
curve,
run
molar
in the
perturbation that it
of
the formatiseems reasona-
in the conditions
of the
to each Hb subunit. probable,
since
That in
the
BIOCHEMICAL
Vol. 85, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
3
Figure 3. Kinetics of the titration curve of the mixture metHb-Cyt b5. The numbers 3 to 15 represent the minutes after the start of the electrophoreprior tic run at which pictures were taken. The same gel was photographed to staining up to the fifth frame. The sixth frame represents the same gel as the fifth, but after staining with Coomassie Blue G-250. All other conditions and symbols as in Fig. 1.
red that
cell
the concentration
of metHb (cu. Fig.
2~10~~
of the two proteins.
Fig.
1. Here,
this
M) under
3 shows the kinetic
ture
better
of Cyt b5 is very
appreciated
from
behavior
This
the pH range
physiological
is
(8x10 -7 M) as compared
in general
deviation
region.
1580
to
conditions.
of the titration
of maximum interaction
the strong
low
agreement
curves with
(pH 8.0-8.3) of both
titration
of the mix-
the data
of
can be even curves
in
BIOCHEMICAL
Vol. 85, No. 4, 1978
As the present of interacting ral
technique
appears
macromolecules,
to be very
we should
like
promising
for
to stress
the
the
study
following
gene-
aspects:
1) by running sible
pH-mobility
to determine
stability
curves
the nature
of the complex
2) equilibrium se complexes.
species,
run
if during
the
since is
half
even
life, will
within
in most cases favorable
of a few minutes,
in a mixture,
species
groups, all
in the most
of the order
to equilibrium transient
protein
of the interacting
focusing
In fact, complexes
of interacting
and its
isoelectric
fe of these
apart
AND BIOPHYSICAL RESEARCH COMMUNlCATlONS
will
it
is
the pH range a single fail
the
have ample
of
experiment;
to detect
cases,
pos-
the-
the half
li-
interacting time
to split
state.
ACKNOWLEDGEMENTS: Supported in part by INSERM (grants CL 37 76 69) and by Consiglio Nazionale delle Ricerche (CNR, 01471.04). P.G.R. thanks INSERM for a visiting professorship tut de Pathologie Moleculaire (Paris). We are grateful to Krishnamoorthy and H. Wajcmann for helpful criticism and
78 5 149 3 and ATP Roma, grant CT 77 to the InstiDrs. D. Labie,R. discussion.
REFERENCES 1.
Rosengren, A., Bjellqvist, B. and Gasparic, V. (1977) in Electrofocu-z sing and Isotachophoresis (Radola, B.J. and Graesslin, D., eds.) de Gruyter, Berlin, pp. 165-171. 2. Righetti, P.G., Krishnamoorthy, R., Gianazza, E. and Labie, D. (1978) J. Chromatogr., in press. 3. Krishnamoorthy, R., Bianchi Bosisio, A., Labie, D. and Righetti, P.G. (1978) FEBS Letters 94, 319-323. 4. Gacon, G., Leroux, A., Lostanlen, D., Labie, D. and Kaplan, J.C. (1978) 12th FEBS Meeting, Dresden, Abstr. No. 826. 5. Hultquist, D.E. and Passon, P.G. (1971) Nature New Biol. 229, 252-254. 6. Omura,T. and Takesue, S. (1970) J. Biochem. (Tokyo) 67, 249-257. P.G. and Drysdale, J.W. (1974) J. Chromatogr. 98, 271-321. 7. Righetti, P.G. and Chillemi, F. (1978) J. Chromatogr. 157, 243-251. 8. Righetti, 102, 563-568. 9. Salemme, F.R. (1976) J. Mol. Biol. 10. Ng, S., Smith, M.B., Smith, H.T. and Millet, F. (1977) Biochemistry 16, 4975-4978.
1581