From the
Five different human hemoglobins were used to test the postulate
that dissociation of hemoglobin (Hb) tetramers into
Hemoglobin (Hb) is the oldest and probably the most thoroughly
studied example of an allosteric protein
(1) . The concept of
``linked functions'' was, in fact, introduced by Wyman almost
50 years ago
(2) in order to analyze the reciprocal relation
between the binding of oxygen on the one hand and that of CO
In the course of
developing a spectrophotometric method for measuring the rate and
equilibrium of heme exchange between Hb and serum albumin, we found
recently
(3) that heme transfer from Hb dimers is very much
faster than that from tetramers. This suggested that binding of heme to
globin and binding of
In this report we present evidence that
this is indeed the case and that measurements of the rate of heme
transfer as a function of the Hb concentration can therefore be used to
determine the tetramer-dimer dissociation constant of normal, mutant,
and chemically modified hemoglobins.
HbA, the major component of normal human blood, was isolated
and stored in liquid nitrogen as described previously
(4) . The
concentration of all hemoglobin solutions was measured
spectrophotometrically after conversion to ferrihemoglobin-cyanide,
using 1.1
Hb Yakima (Asp
HbA,
carboxymethylated at all four N-terminal residues
(
Des-Arg hemoglobin was prepared as described by
Kilmartin
(9) , except that we used 0.1 M barbital
buffer for the carboxypeptidase digestion instead of 0.2 M.
Human serum albumin and Tris buffer were purchased from Sigma, and
the former was used to prepare methemalbumin as described
(3) .
The hemoglobins were oxidized to the ferric form with 1.2
equivalents of potassium ferricyanide at room temperature, followed by
removal of ferro- and ferricyanide with Sephadex G-25
(3) .
The initial rate of heme transfer from ferrihemoglobin
(Hb
We used a Hewlett-Packard diode
array spectrophotometer (model 8451A) to follow the progress of the
heme transfer. This instrument is especially convenient for this
purpose because it has a built in program for multicomponent analysis
in an ``overdetermined'' system, i.e. when the
number of data points exceeds the number of components
(11) . For
other spectrophotometers, measurements at two wavelengths and solution
of the simultaneous equations, relating the absorbance to the
concentration and extinction coefficients of the two components, can be
used. Detailed directions are given in Ref. 12.
All the measurements
of the rate of heme transfer were done in 0.25 M Tris buffer
at pH 9.0 at 20.0 °C with equal initial concentrations of human
serum albumin and Hb
A more than 3000-fold range of Hb concentration could
be covered by varying both the length of the light path (from 1 to 50
mm) and the wavelength interval for the measurements (380 to 420 nm for
the low and 470 to 630 nm for the higher concentrations).
It should
be stressed that these measurements of the initial rate of heme
transfer involve only the
The relation between the first order rate constant,
k, and the Hb
On-line formulae not verified for accuracy Alternatively, the linearized form of Equation 1 may be used,
i.e. log (1 -
The concordance of the two sets of constants in
further suggests that neither the pH (9.0 for our
measurements and 7.4 for those of Turner et al.(8) )
nor the oxidation state of the iron (Fe
Finally, it should be emphasized that the
linkage we have described between dissociation of heme from globin and
dissociation of
We thank Dr. Sam Charache and Dr. Robert Koler for
generous donations of blood samples containing Hb Osler and Hb Yakima,
respectively, and Rini Kwan for determining the pK of the hemoglobins
in this laboratory.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
dimers
and dissociation of heme from globin are linked reactions.
Spectrophotometric measurements of the initial rate of heme transfer
from Hb to serum albumin were made over a 3000-fold range of Hb
concentration and yielded the heme-globin dissociation rate constant
for tetramers and that for dimers. The tetramer-dimer dissociation
constant (K
) could then be calculated from the rate
constant at intermediate concentrations. The values obtained for the
five hemoglobins, spanning a 250-fold range in K
, were in
good agreement with those found by direct methods. The relation between
this new linkage reaction of hemoglobin and the classical ones, such as
the reciprocal relation between the binding of oxygen and protons, is
discussed briefly.
or protons on the other, by hemoglobin.
dimers to each other are yet another
example of linked functions.
10
as the molar extinction coefficient at
540 nm (heme basis).
His)
and Hb Osler (Tyr
Asp) were isolated from the
blood of patients, heterozygous for these mutants, as
described
(5, 6) , except that fast protein liquid
chromatography on Q-Sepharose was used for Hb Yakima, and
DEAE-Sepharose chromatography with a linear gradient of 50 mM
Tris buffer from pH 8.35 to pH 7.00 was employed for Hb Osler.
) was
prepared according to DiDonato et al.(7) with the
following changes: only a 20-fold molar excess of sodium
cyanoborohydride was used for the reduction, and the initial pH for the
DE-52 cellulose separation was 7.7 instead of 8.3.
(
)
) to human serum albumin was measured
spectrophotometrically as described earlier
(3, 10) .
This method is based on the fact that the large spectral change in
ferrihemoglobin, due to the ionization of its iron-linked water
molecule, is absent in methemalbumin
(3) . The pK of this
transition was therefore first measured spectrophotometrically for all
the hemoglobins used. The two mutant hemoglobins as well as the two
chemically modified ones were found to have the same pK as HbA,
i.e. 8.0 ± 0.1.
. Since these reactions involve
the transfer of heme from tetramers and dimers (Hb
) to
a monomer (human serum albumin), all Hb
concentrations
are given on a heme basis and are listed as micro-normal
(µN) to indicate this fact. The reaction rate was first
order with respect to [Hb
] up to about 20%
heme transfer, and the rate was independent of the albumin
concentration.
chain hemes, since the ones on the
chains are bound much more tightly and released much more slowly
(13-16).
concentration for three of the
hemoglobins is shown in Fig. 1. It is clear that these data
permit the determination of both the rate constant for dimers,
k
= 0.12 min
, and that
for tetramers, k
= 0.006 min
by simple extrapolation.
Figure 1:
The rate of
heme transfer as a function of the Hb concentration.
The rates were measured at 20 °C in 0.25 M Tris, pH 9.0,
with initial [Hb
]/[HSA] =
1.0, as described under ``Materials and Methods.'' Filledcircles, HbA; opentriangles,
(
);
opensquares, Hb Yakima.
The tetramer-dimer dissociation
constant, K, may then be calculated at any
intermediate concentration from the rate constant at that
concentration; if k
is the rate constant at
concentration c (heme basis) and
is
the fraction of hemes present as dimers, then
= (k
-
k
)/(k
-
k
). Since [D] =
/2 and [T] = (1 -
)
/4, then,
) - 2 log
=
-log K + log c. The value of log K is then obtained from the intercept at (log (1 -
)
- 2 log
) = 0. The data for the five hemoglobins are
plotted in this way in Fig. 2, and the values of K
derived from these plots are compared with those reported by
Turner et al.(8) in .
Figure 2:
Determination of K.
Conditions for the measurements were as in Fig. 1. K
= [D]
/[T] is the value of
[Hb]
when log (1-
) = 2 log
. Opensquares, Hb Yakima; opencircles, des-Arg Hb; filledcircles,
HbA; filledsquares, Hb Osler; opentriangles,
(
).
It is clear from
these data that our values for the tetramer-dimer dissociation constant
of these five hemoglobins, which span a 250-fold range in the numerical
value of K, are in very reasonable agreement with those
reported by Turner et al. (8), using a much more complicated
and demanding methodology. Measurement of the initial rate of heme
transfer from ferrihemoglobin to serum albumin as a function of the Hb
concentration is therefore a very simple way to determine the
tetramer-dimer dissociation constant of liganded hemoglobin and should
be a convenient way to screen mutant hemoglobins for alterations in
this parameter.
in our
measurements and Fe
in those of Turner et
al.(8) ) can have an important effect on the tetramer-dimer
dissociation constant.
dimers from one another differs in a
fundamental way from the classical linkage reactions of hemoglobin. The
latter are linkages between the affinities, i.e. the
equilibrium reactions of different ligands, whereas here we are dealing
with the relation between the rate of dissociation of heme
from globin and the equilibrium dissociation of the tetramer.
Table:
Tetramer-dimer dissociation constants
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.