©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Coupled Reactions in Hemoglobin
HEME-GLOBIN AND DIMER-DIMER ASSOCIATION (*)

Ruth E. Benesch (§) , Suzanna Kwong

From the (1) Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons of Columbia University, New York, New York 10032

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Five different human hemoglobins were used to test the postulate that dissociation of hemoglobin (Hb) tetramers into 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.


INTRODUCTION

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 or protons on the other, by hemoglobin.

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 dimers to each other are yet another example of linked functions.

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.


MATERIALS AND METHODS

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 10 as the molar extinction coefficient at 540 nm (heme basis).

Hb Yakima (Asp 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.

HbA, carboxymethylated at all four N-terminal residues () 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.()

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) 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.

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. 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.

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 chain hemes, since the ones on the chains are bound much more tightly and released much more slowly (13-16).


RESULTS AND DISCUSSION

The relation between the first order rate constant, k, and the Hb 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,

On-line formulae not verified for accuracy

Alternatively, the linearized form of Equation 1 may be used, i.e. log (1 - ) - 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.

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 in our measurements and Fe in those of Turner et al.(8) ) can have an important effect on the tetramer-dimer dissociation constant.

Finally, it should be emphasized that the linkage we have described between dissociation of heme from globin and dissociation of 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



FOOTNOTES

*
This work was supported by National Institutes of Health Merit Award HL-05791. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Tel.: 212-305-3891; Fax: 212-305-7932.

J. M. Manning, personal communication.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.