The Effect of 2,3=Diphosphoglycerate on the Tetramer-Dimer ...On this basis Gibson suggested that alkaline form of hemo- globin, designated IIb*, represents a transient form of deoxyHb

THE JOURSAL OF B~OLOGKAL CHEMISTRY Vol. 249, No. 9, Issue of May 10, PP. 2879-2885, 1974

Printed in C.S.A.

The Effect of 2,3=Diphosphoglycerate on the

Tetramer-Dimer Equilibrium of Liganded

Hemoglobin*

(Received for publication, September 26, 1973)

ROBERT D. GRAY

From the Department of Bioclremistrg, ll~liversity of Louisville School of dletlicine, Health Sciences Center, Louisville, Keducky /,0201

SUMMARY

The effect of 2,3-diphospho-D-glycerate (Pg-glycerate) on the kinetic behavior of deoxyhemoglobin generated by the rapid dissociation of gaseous ligand (either by flash photolysis of carbon monoxide hemoglobin (HbCO) or by deoxygena- tion of oxyhemoglobin (HbO,) in the presence of sodium dithionite) is consistent with a stabilization of the tetrameric state of liganded hemoglobin by the organic phosphate.

The fraction (a) of rapidly reacting hemoglobin produced by pulsed laser photolysis of phosphate-free HbCO in 0.05 M 2,2’-bis(hydroxymethyl)-2,2’,2”-nitriloethanol-O.l M NaCl, pH 7.0, was independent of CO concentration below about 500 pM, but increased systematically with dilution of the hemoprotein. The apparent tetramer-dimer dissociation constant (Kt,2) calculated from the dependence of (Y on [HbCO] was 3.6 f 1.0 pM in the absence of phosphates and decreased to 1.4 rt 0.3 PM when 1 mM Py-glycerate was added. These values of Kt,2 are similar to estimates for liganded hemoglobin obtained in sedimentation experiments conducted at pH 7.0 in 0.1 M phosphate (EDELSTEIN, S. J., REHMER, M. J., OLSON, J. S., AND GIBSON, Q. H. (1970) J. BioZ. Chem. 245, 4372-4381) and in 0.1 M Tris-0.09 M NaCl (KELLETT, G. L. (1971) J. Mol. Biol. 59, 401424).

The magnitude of the Soret absorbance drift accompanying deoxygenation of dilute solutions of HbOL in the presence of sodium dithionite was decreased by added 1 mM P2-glycerate, and the second order rate constant characterizing the drift phase was increased at 20” from 0.54 f 0.09 PM-’ s-l to 1.57 f 0.18 PM-’ s-l by the organic phosphate. Since the drift

has been shown to result from the formation of deoxy tetra- mers from deoxy & dimers (KELLETT, G. L., AND GLTT- FREUND, H. (1970) Nature 227, 921-926), this result is also consistent with the proposed phosphate inhibition of dimer formation by liganded hemoglobin.

In 1934 Roughton (1) reported that deoxyHb’ produced by dissociation of oxygen from HbOz in the presence of sodium

* This work was supported by National Science Foundation Grant (;B-32283.

1 The abbreviations used are: deoxyHb, deoxyhemoglobin;

dithionite maintains under certain conditions an anomalously high reaction rate with CO for a brief time. Since then a number of so-called “rapidly reacting” species of hemoglobin have been described, all of which are characterized by an increased ligand affinity and a slightly decreased Sorct absorption whose mas- imutn is redshifted apprositnatcly 2 nm (2). In each case the presence of the high affinity material can presutnably be traced to the transient or pertnanent existence of deoxyHb in the ligand- bound confortnation. For example, Ivhen the amount of CO removed from HbCO by flash photolysis is restricted to about 554, the rate of CO recombination at pH 7 is 3 ~1r-l s-l com- pared to an initial rate of about 0.4 ~~1-l s-l when total photol- ysis is achieved (3). These two rate extremes have been as- cribed to CO binding to the intermediate species Hb,(CO)$ and to Hbr, respectively, and thus probably reflect structural dif- ferences (3, 4) between the ligand-bound and ligand-free con- formations of the protein.

h second form of rapidly reacting hemoglobin was observed by Gibson (5) when Hash photolysis of sheep HbCO was carried out at low temperature and alkaline pI-I. The proportion of rapid recombinant increased in this instance with increased CO concentration, but was independent of IIbCO concentration. On this basis Gibson suggested that this alkaline form of hemo- globin, designated IIb*, represents a transient form of deoxyHb which exists in the high affinity, ligand-bound conformation for a short period after photolysis.

h third form of quickly reacting hemoglobin appears at low hemoglobin concentration (6, 7). Recent experiments cor- relating sedimentation and kinetic data have shown that the rapidly reacting component produced by dilution at neutral pH in the concentration range 0.1 to 100 PM is the liganded C@ dimer (8).

The effect of removing endogenous phosphate compounds (9-11) on the kinetics of hemoglobin recombination with CO after Nash photolysis has been mentioned by several investigators. Gibson and Parkhurst (12), Gray (13), and MacQuarrie and Gibson (14) observed relatively large amounts of rapid hemo- globin when stripped HbCO was photolyzed. It is obviously important to elucidate the structural basis for this increased proportion of rapidly reacting material which appears with

P*-glycerate, 2,3-diphospho-n-glycerate; bis-tris, 2,2’-bis(hy- droxymethyl)-2,2’,2”-nitriloethanol; HbCO, carbon monoxide hemoglobin; HbO,, oxyhemoglobin.

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stripped HbCO, both in order to interpret the kinetics and also to appreciate fully the role of organic phosphates in hemoglobin- l&and equilibria. The results of the kinetic experiments re- ported here (subject, of course, to the inherent inability of ki- netic data alone to define unambiguously chemical structures) suggest that stripped, liganded hemoglobin dissociates to rapidly reacting dimers to a greater degree than when Pz-glycerate or inorganic phosphate is present.2

The following reaction scheme may facilitate presentation and discussion of the results.

kf 2 b(co)P(co)l2 f 2~(CO)P(CO)

II

ki.4 hv k,

k” hv k,

IT 4co + [apI* 4, 4,2 ’ 2ap + 4co

kF.4

In the scheme, A$,2 and ky,2 are the rate constants for forma- tion of dimers from liganded and unliganded tetramers, respec- tively; ki,q and ky,, are the corresponding rate constants for the dimer-dimer association reaction. The pseudo-first order rate constant for CO binding to qB dimers is k, and that for the slower binding of CO to tetramers is k,. A CO concentra- tion is used so that insignificant adjustment of the tetramer- dimer equilibrium occurs during the cyclic displacement of bound CO by the flash and the reassociation dark reaction. The equilibrium constants for tetramer-dimcr dissociation of liganded and unliganded hemoglobin are I<:, 2 and KY, 2, respec- tively, where Kt,, >> Ky,2 (8, 16).

EXPERIMENTAL PROCEDURE

Materials

Preparation of Hemoglobin-IIemolysates were prepared from fresh human blood by the method described bv Gibson (17), with the exception that stroma were removed by Fentrifugation’ after the addition of 0.25 volume of 5% NaCl. Polvacrvlamide elec- trophoresis of the resulting hemoi;sate in 5% gkls at pH 9.5, fol- lowed by staining with Coomassie blue R250 in 10% trichloroacetic acid, showed a major band corresponding to HbA and two minor components.

Small molecules were removed by gel filtration of the hemolysate using Sephadex G-25 equilibrated with 2.5 rnM bis-tris-0.1 r,l NaCI, pH 7.4 to 7.6.

Aliquots of t,he resulting stripped hemoglobin (at least 0.1 rmole of IIbr) were analyzed for t,otal phosphate by method of Ames (18) as modified by Gray and Gibson (19). In most cases no phos- phate could be detected after gel filtration; sometimes traces of phosphate remained which were the equivalent of approximately 3 to 4% of the hemoglobin tetramers.

Working solutions were prepared by diluting the stock hemoly- sate (2 to 4 mM heme equivalents) directly into the desired buffer solution. Hemoglobin concentrat,ion was estimated spectro- photometrically using the extinction coefficients of Bannerjee et al. (20).

&ageTlts-Bis-tris was obtained from Aldrich Chemical Co., Milwaukee, Wis., or from Sigma Chemical Co., St. Louis, MO. Pg-glycerate was obtained from Sigma as the pentacyclohexylam- monium salt; stock solutions of approximately 10 mM were pre- pared as described by Benesch et al. (11) and analyzed to deter- mine the exact phosphate concentration (19). CO was a product of Matheson Co., Joliet, Ill., and prepurified N, was obtained from Air Products and Chemicals, Inc., Emmaus, Pa. Solutions con- taining CO were prepared by mixing, in the desired proportions, buffers previously equilibrated with water vapor-saturated CO or N,. The solubility of CO was taken to be 1.0 mM at 20” and at- mospheric pressure (21). Sodium dithionite (Mannox Brand)

2 While this work was in progress, an abstract of light-scattering experiments was published which indicates that Pz-glycerate inhibits dimerization of liganded hemoglobin (15).

was included in t,he buffers used in photolysis at a concentration of 0.01%.

The dithionite solutions for deoxygenation of HbOx were pre- pared by adding a weighed quantity of the solid salt to deoxy- genated 0.05 M bis-tris 0.1 M NaCl, pH 7.0, in an evacuated tonom- eter. The pH of the buffer was unchanged after the dithionite addition.

Methods

Stopped Flow Ezperiments-Deoxygenation of HbO, was carried out by mixing air-equilibrated hemoglobin solutions (0.05 M bis- tris-0.1 M NaCl, pH 7.0) with 0.4% sodium dithionite in a stopped flow spectrophotometer of the Gibson-Milnes design (22). A Durrum grating monochromator was used to obtain monochro- matic light. The half-bandwidth was 1.5 nm and the temperature was maintained at 20 =t 0.2”. The dead time of the instrument was 2.5 ms.

Photolysis Experiments-Photolysis of HbCO was accomplished by illuminating the sample in an airtight plexiglass cuvette (3 x 3 X 3 mm) with a pulse of coherent radiation from a flashlamp- pumped organic dye laser (Chromabeam model 1050, Synergetics Research, Inc., Princeton, N.J.). The organic dye used was 0.1 mM rhodamine 6G in either ethanol or methanol. Broadband optics were utilized to produce a pulse of approximately 250 mJ in 650 ns centered at approximately 590 nm.

The kinetics of CO iecombinatihn was measured in 0.05 M bis- tris-0.1 M NaCl, pH 7.0, at 20 f 2”, by observing the change in light transmission of the hemoglobin solution in a direction at riaht angles to the photolysis source. The spectroscopic measur:ng system consisted of a high intensity source (Osram XBO 150 W/l xenon arc lamp or a 150-watt quartz-halogen lamp)3 powered by a stabilized power supply (23). Radiation from the source was collimated and passed through heat-absorbing filters, a blue filter (Ealing Optical Co., No. 26-3376. Chance No. 0.13.10). the observa- tion c&,-and a grating monochromator (EU-700/E, Heath Co., Benton Harbor, Mich.). The half-bandwidth was 1.5 nm. The output current of the lP28 photomultiplier was transformed to a voltage by an operational amplifier (Analog Devices 405) with a I-MQ precision feedback resistor. The time constant of the sys- tem could be varied upwards from 1.5 ps; values of 0.03 or 0.04 ms were routinely employed.

Data Collection-Transmittance data from both stopped flow and photolysis experiments were collected automatically with a 12.bit analog-digital converter (Analog Devices ADC-12 Q) inter- faced to a digital computer (Data General Nova 1200) in a system similar to that designed by DeSa (24). The system was checked for reliability by measuring the kinetics of CO combination with sperm whale myoglobin over a range of CO and myoglobin con- centrations.

Data collection for the photolysis experiments was initiated 10 ps after firing the laser. Th e photomultiplier voltage was sampled at two rates differing by a factor of 10 in order to ac- curately represent the entire biphasic recombination. A reference voltage was ascertain 1 s after collection of the final data point.

The normalized absorbance data AA,/AA, were fit by a least squares minimization technique (25) to Equation 1:

AA,/AA, = Fe-k/’ + (1 - F)e-ksl (1)

where F is the fractional amount of the rapid species present at time (t) = 0 and kf and k, represent the apparent first order rate constants for the reaction of the rapid and slow species with CO. F, kf, and k, were treated as adjustable paramet’ers and the best value of each obtained by minimizing the error in the normalized absorbance changes using the normal equations for F, kf, and k,. The standard deviation of the parameters was determined from the error matrix. In all cases at least a 5-fold excess of CO was present in order to assure the approximate pseudo-first order requirement for CO and Hb combination. Computations were carried out using programs written for the Nova 1200 computer.

The experimental protocol for the stopped flow deoxygenation experiments depended on which reaction was of interest. Data collection was initiated 3 ms after flow-stopping switch for the deoxygenation reaction. When the kinetics of the slow absorb- ance changes subsequent to deoxygenation was to be studied, data

3 The intensity of the measuring light beam did not significantly alter the results; see Table I.

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collection started 100 ms after flow stopping. A reference voltage was collected 30 or 60 s later. This procedure had the advantage that by omitting observation of the initial deoxygenation (AA N 1) reaction, a greater sensitivity could be used to observe the slow reaction (AA N 0.1). Estimation of the actual absorbance change in the slow phase was difficult because: (a) at 429 nm the slow changes amounted usually to less than 10% of the total AA; (5) the slow second order reaction followed a first order reaction which made it impossible to establish accurately the “zero” time for the second order reaction; (c) occasional artifacts resulting from dithionite were experienced. The requirements adopted for using a particular data set were: (a) the series must show linear l/AA versus time plots over 90% of the total AA at 429 nm; (b) no detect- able difference in the AA measured whether the final reference voltage was collected 30 or 60 s after the last data point. An esti- mate of AA due to the slow second order reaction was obtained by extrapolating plots of the reciprocal of the absorbance change versus time to zero time.

RESULTS

Laser Photolysis of NbCO-The kinetics of recombination of stripped human Hb subsequent to photolysis in the presence and absence of Pz-glycerate is illustrated in Fig. 1. The kinetics was measured at 437 nm, a wavelength isosbestic for both the rapidly and slowly reacting species. In the absence of phos- phates the rapid species contributed 32.5 + 0.4y0 to the ob- served absorbance change; when the sample was supplemented with 623 PM Pz-glycerate, the percentage of rapid material de- creased to 25.3 f 0.6%. This analysis can, at best, be only approximate since it is well known that the second order rate constant for CO binding to Hb depends upon the fractional saturation with ligand (26). However, it does serve to point out the effect of P2-glycerate. In experiments shown in Table I, 0.1 M phosphate also depressed the proportion of rapidly reacting hemoglobin to about the same extent.

Fig. 2 shows that the fraction of rapid material depends on the wavelength of the observing light. The lines are calculated from the data in Fig. 2a of Cassoly and Gibson (27), assuming that the rapid species possesses the HbCO-deoxyHb difference

1.0

0.8

0.6

0.5

0.4

0.3

0

a” \ 0.2

:

0.10

0 08

0.06

o.0401 40 60 80 100

TIME (ms)

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spectrum of the isolated (Y and p chains and the slow species has the HbCO-deoxyHb difference spectrum of cooperative hemo- globin tetramers. The experimental points of Fig. 2 show that the spectral characteristics of the rapidly and slowly reacting species compare favorably with that expected if the unliganded rapidly reacting species has the chain like HbCO-dcoxyHb dif- ference spectrum characteristic of noncooperative hemoglobin derivatives.

The relationship between HbCO concentration and the frac- tion of rapid reaction, (Y, is shown in Fig. 3 for stripped hemo- globin (open circles) and for hemoglobin in the presence of PZ- glycerate (@filled circles). A value of Kt, 2 was calculated for each data point in Fig. 3 using the expression or*[HbCO]/(l - ar) ; average values for Kt,2 of 3.6 f 1.0 PM for stripped and 1.4 f 0.3 PM for hemoglobin with Pz-glycerate were obtained. The agreement between the behavior observed and that predicted on the basis of the dimerization scheme suggests that the rapidly reacting hemoglobin observed subsequent to flash photolysis of stripped HbCO can be accounted for entirely by assuming it to be the ap dimer.

TABLE I Photolysis of carbon monoxide hemoglobin

Conditions: [Hb] = 10.2 pM; [CO] = 100 pM; 0.05 M bis-tris-0.1 M NaCl, pH 7.0, 23 =t 2”.

Experi- ment Addition

None None” 0.1 M phos-

phate 0.1 M phos-

phate* 1 rnM Pz-

glycerate

Fraction of Nb

reacting rapidly

0.447 0.448 0.318

0.323

0.343

k/ ks

&&f-l s-1

6.15 f 0.35 0.28 f 0.08 5.69 f 0.45 0.29 f 0.01 5.50 f 0.02 0.23 f 0.00

5.69 i 0.02 0.23 f 0.00

5.73 f 0.37 0.21 f 0.00

-

-

K4.2

PM 3.70 3.70 1.55

1.60

1.90

a Intensity of measuring light reduced b.y 47yo compared to Experiment 1.

* Bis-tris and NaCl omitted.

t I I I I

410 420 430 440 450

Wavelength (nm) FIG. 1. Normalized kinetics of the recombination of CO with deoxyHb subsequent to the laser photolysis of stripped human FIG. 2. Dependence of the fraction of stripped deoxyHb react- HbCO. Conditions: [CO] = 100~~; [Hb] = 14.5~~, [Pz-glycerate] ing rapidly with CO after photolysis on the wavelength of the = 0 (O---O); [Hb] = 13.5 PM, [Pz-glycerate] = G23 PM (O--O); measuring light. Conditions: [Hb] = 13.6 NM; [CO] = 100 PM; 0.05 M bis-tris-0.1 M NaCl, pH 7.0, 21 f 2”. 0.05 M bis-tris-0.1 M NaCl, pII 7.0,21 f 2”.

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o~500.10.2 [2,3- Diphosphoglycerate] (mM)

FIG. 4. Dependence of the fraction of deoxyHb reacting slowly with CO subsequent to photolysis of HbCO on Pn-glycerate con- centration. Conditions: [CO] = 100~~; [Hb] = 9.OpM (O--O); [Hb] = 15.0 PM (O-O); 0.05 M bis-tris-0.1 M NaCl, pH 7.0, 21 f 2”.

FIG. 3. Fraction of deoxyHb reacting rapidly with CO subse- quent to laser photolysis of stripped HbCO. 0-0, no phos- phates; O-0, 0.6 to 1.5 rn~ Pn-glycerate; 0.05 M bis-tris-0.1 M NaCl, pH 7.0, 21 f 2”.

An approximate equilibrium constant for binding of PZ-glyc- erate to stripped HbCO in 0.05 M bis-tris-0.1 M NaCl, pH 7.0, can be obtained by observing the fraction of photolytically produced rapid hemoglobin as a function of Pz-glycerate con- centration (Fig. 4). The concentration of Pz-glycerate required to give half the maximal decrease in rapid hemoglobin is in the range 100 to 300 PM. This relatively high concentration reveals that the interaction is weak compared to phosphate binding by deoxy Hb (28). The difference in the two curves of Fig. 4 was not investigated further but may result from the use of different hemoglobin preparations.

The effect of CO concentration on the fraction of rapidly re- acting stripped hemoglobin was also tested. Increasing CO from an excess of 5-fold to nearly 30.fold had only a small effect on cr (Table II). This experiment indicates that a first order conformational change which converts rapidly to slowly re- acting hemoglobin after the photolytic flash does not become rate-limiting in the absence of phosphates in the range of CO concentrations tested.

Deoxygenation of HbOz-Gibson and Roughton (discussed in Ref. 3) noted that slow absorbance changes accompany the rapid deoxygenation of HbOz by sodium dithionite under certain carefully controlled conditions. The magnitude of this so-called “drift phase” depends on the wavelength of observation and also on the concentration of HbOz (28, 29). Kellett and Gutfreund (29) correlated the size of the drift phenomenon with the fraction of oxygenated crp dimers (determined by sed- imentation equilibrium (30)) in 0.01 M Tris-0.09 M NaCI-1 mM EDTA, pH 7.0. Their results suggested that the absorbance changes following the deoxygenation process were the result of the relatively slow (ky,, = 0.43 FM-’ s-l) association of the unliganded dimers produced by deoxygenation of liganded hemo- globin in dilute solution. The effect of phosphates on the mag-

Dependence of fraction of rapidly reacting stripped hemoglobin on [CO]

Photolysis of 14.7 pM stripped HbCO in 0.05 M bis-tris-0.1 M NaCl, pH 7.0. The temperature was 21 f 2”. The rate constants are the apparent first order constants estimated by least squares analysis of the average of at least four successive experiments as described in the text.

ICOI Fraction rapid hemoglobin (a) k/

I ks

/JJf

105 205 316 516 802

0.413 0.373 0.423 0.433 0.470

s-1

Fl6 25.3 1340 50.8 2005 72.5 3630 112.7 4045 166.4

sequently, this author repeated the deoxygenation experiments using stripped HbOz in the presence and absence of Pz-glycerate.

Figs. 5 and 6 show the wavelength dependence of the deoxy- genation reaction of stripped HbOz by dithionite. Fig. 5 il- lustrates that the apparent rate of deoxygenation of stripped HbOz progressively decreases toward the end of the reaction when observed at 429 nm. In contrast, no such deceleration is found when the reaction is followed at 437 nm. This result shows the absence of dithionite-dependent artifacts within the time range of these experiments (-60 s).

Fig. 6 shows that the kinetic difference spectrum of the slow phase after HbOz deoxygenation exhibim the characteristics ex- pected of conversion of a noncooperative derivative to a co- operative form of unliganded hemoglobin (2).

When the magnitude of the absorbance change due to the slow phase is plotted as a function of the initial concentration of HbOt, the results of Fig. 7 are obtained. In Fig. 7 the lines corresponding to stripped and Pz-glycerate-supplemented solu- tions of hemoglobin arc the theoretical ones based on A~gr$~“’ = 12 (determined by the method of Kellett and Gutfreund (29) in 1 M NaI) and 1<t,2 = 3.6 PM for stripped and 1.4 pM for phos- phate-liganded hemoglobin. The results of the deoxygenation

nitude or the kinetics of the drift phase was not reported. Con- experiments are thus consistent with the interpretation of the

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0.6

0.5

0.4

0.2 - 02 - 8 8

5 5

Lf Lf

i? i? m m clo.1 - a01 -

a a 0.06 - 006 -

0.07 - 007 -

0.06 - 006 -

0.05 - 005 -

0.04 - 004 -

0.02’ 0 20 40 60 80 100

TIME (ms)

FIG. 5. Kinetics of stripped HbOz deoxygenation by sodium dithionite measured at 429 or 437 nm. The apparent first order rate constant at 50% saturation was 31.3 s-1 at 437 nm. Condi- tions: [HbOz] = 10.0 PM; [Na&ItOa] = 0.4% before mixing; 0.05 M bis-tris-0.1 M NaCl, pH 7.0, 20”. Reference voltage was collected 60 s after mixing.

0.06

w

g 0.02 Q m fK cl

ii 0

Q

a I -0.02

-0.04 - I I I I I I I

410 420 430 440 450

x (nm)

FIG. 6. Kinetic difference spectrum obtained from the slow phase after deoxygenation of stripped Hb0.r by sodium dithionite. Conditions: [HbOl] = 6.0 PM; [Na&03] = 0.4% before mixing, 0.05 M bis-tris-0.1 M NaCl, pH 7.0, 20”. FIG. 8. Second order kinetic plot of the drift reaction observed

after deoxygenation of HbO, by dithionite. Conditions: [Na&%Oa] = 0.4%; stripped [HbOt] = 6.0 pM (0-O) or 12.0 PM (O-0) in 0.05 M bis-tris-0.1 M NaCl, pH 7.0; [HbOn] = 6.8 /.IM (A---A) or 2.6 PM (A-A) in 0.05 M bis-tris-0.1 M NaCl-1 mM

Pz-glycerate, pH 7.0; [HbOz] = 15.9 pM (It-m) in 0.05 M bis- tris-0.1 111 NaCl-1 M NaI, pH 7.0. HbOs and dithionite concentra- tions are given before mixing. Temperature = 20”.

flash photolysis experiments: phosphates apparently reduce the extent of dimer formation in solutions of liganded hemoglobin.

The kinetic data of Fig. 8 are presented as additional evidence supporting this conclusion. When the reciprocal of the ab- sorbance change at 429 nm is plotted as a function of time after mixing with dithionite, a linear relationship is obtained over at least 85ya of the drift reaction. Such kinetic behavior, in- dicative of a second order reaction of the type 2A - B, is most reasonably interpreted as the association of unliganded dimers to give cooperative unliganded tetramers. The experiments with the value of Ae given above, were 0.54 f 0.09 pMel s-l

illustrated in Fig. 8 also show that Pz-glycerate increases the rate of dimer association. The bimolecular rate constants calculated from the slopes of a series of such experiments, in conjunction

.20

.I6

0 0 4 8 I2 I6

FIG. 7. Magnitude of the slow absorbance change subsequent to deoxvaenation of strioned HbO, bv 0.4% sodium dithionite in the presence and absence- of Pz-glycerate:‘ Hemoglobin concentra- tions are before mixing; the lines are calculated based on: K& = 60 FM in 1 M NaI (0-O) (29); 3.6 PM for stripped HbOz (O--O); and 1.4 KM for 1 mM Pn-glycerate (A-A); 0.05 M bis-tris-0.1 M NaCl, pH 7.0, 20”.

0 IO 2.0 30 40

TIME 1s)

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(nine experiments, [HbOz] = 2.1 to 17.2 pal) and 1.57 ZIZ 0.18 /.LM-~ S-l (eight experiments, [HbOn] = 1.3 to 17.6 PM) for stripped and P2-glycerate-supplemented hemoglobin at 20”, respectively. Note also that values of the intercepts are inversely related to the initial HbOz concentration and are decreased in magnitude by the dissociating agent NaI and increased by Pi-glycerate.

DISCUSSION

The liganded, or R allosteric conformation of hemoglobin (31, 32) binds CO at a rate about 80 times that of the 2’ or deox) conformation. Of the several mechanisms discussed in the introductory section of this paper in which phosphate com- pounds might influence the transition between the high and low affinity forms subsequent to ligancl removal, the data suggest that the primary mechanism is a stabilization of the liganded tetrameric structure relative to the dimer in the presence of phosphates.

The dependence of (Y on [HbCO] (Fig. 3) follows the relation- ship predicted on the assumption that dilution of the protein solution results in dissociation of slowly reacting tetramers to rapidly reacting dimers. The value of k’t,Z calculated from (Y and [HbCO] is 3.6 f 1.0 PM for stripped HbCO in 0.05 M bis- tris-0.1 M NaCl, pH 7.0. This value of Kt,2 is significantly different from that of 1.5 PM estimated by Eclelstein et ~2. (8) from photolysis and sedimentation data obtained with uw

stripped HbCO in 0.1 M phosphate, pH 7.0, and closer to the value of 2.9 PM determined by Kellett (30) for stripped HbOn using the sedimentation equilibrium method and slightly dif- ferent solution conditions (0.1 M Tris-0.09 M NaCl-1 rnhl EDTA, pH 7.0). The data presented in Figs. 1 and 3 and Table 1 show that 1 rnM I’*-glycerate added to stripped HbCO decreased the K ,“,Z to values close to those observed earlier (8) in phosphate buffers.

The relatively high concentration of I’*-glycerate required to achieve half the maximum phosphate effect (about 250 pM, Fig. 4), taken with the fact that P2-glycerate equilibrates rapidly with deoxyHb (18, 33, 34) also is consistent with the importance of an interaction between the phosphate compound and liganded, rather than ligand-free hemoglobin. The association constant of Pr-glycerate and HbCO must be about 4 x lo3 M-I (assuming one binding site per tetramer? as judged from the data in Fig. 4. This value is consistent with equilibrium dialysis data of Cald- well and Nagel (35).

The relative independence of (Y with respect to [CO] eliminates the possibility that phosphates increase the rate of a first order R --t T transformation. Although the data of Fig. 8 show that the rate of deoxydimer association is increased by Pz-glycerate, the increase is not so large that a significant fraction of dimeric deoxyHb could associate to form slowly reacting tetramers prior to CO binding. For example in 1 rnhf Pz-glycerate, the first half-life for dimer association would be approrimately 60 ms when [HbCO] = 35 pM, whereas the half-time for CO binding to the rapidly reacting dimer was less than 10 ms.

The effect of P2-glycerate on the drift phase following di- thionite-induced deoxygenation of gtripped HbOs also supports the conclusion that liganded hemoglobin is stabilized by phosphate compounds. The reduction in the extent of the drift phase by Pz-glycerate (Figs. 7 and 8), the dependence on [HbOz], and the apparent second order kinetics all are best interpreted by as-

suming that Pzmglycerate reduces the eh?ent of dimerization of HbOn.

Osmotic pressure clata of Guidotti (36) indicate that Kt,2 depends to some extent on the nature of the ligand bound to the iron atom of the hemoglobin. In 2 M iYaC1, at pH 7.0 and 20”, the difference in the free energy of tetramer dissociation be- tween HbCO and IIbOz is approximately 300 cal per mole. The values of Kf,2 obtained by the photolytic ant1 deoxygenation methods do not reflect such a difference between the two liganded forms under the conditions of these experiments, since values of 3.6 and 1.4 par for Kf’,2 seem to represent both sets of data adequately (Figs. 3 and 7).

Thomas and Edelstein (16) used Wyman’s (37) linkage equa- tions to calculate that the tetramer-climcr dissociation constant for unligancled hemoglobin should be about 4 x 10-l’ M in 0.1 M phosphate, pH 7.0. Their determination of Ky,2 in this solvent by a titration method gave a value of 3 X lo-l2 M. hp- plication of these linkage equations to stripped deoxyHb in 0.05 M bis-tris-0.1 M NaCl yields a value of 2 x low9 M for KY, 2, assuming that the median ligand activity for oxygen (16, 37) is about 5 PM (38). Thus phosphate compounds may provide a stabilization free energy of about 3.6 Cal per mole at 20” for tetrameric deosyHb compared to the dimeric species.

The kinetic basis of the Pz-glycerate stabilization of deoxyHb tetramers must primarily be an effect of the phosphate on the dissociation rate constant for dimer formation. *issuming the validity of the above estimation of KY,? for stripped deosyHb, the dissociation rate constant ky,2 is approximately 1000 X 1OV s-l. III contrast, the dissociation rate constant in the presence of phosphates would be only 5 x 1OF 0. Thus the rate of association of tleoxy hemoglobin dimers to form tetramers is enhanced by a factor of 2.9, whereas the rate of dissociation to form dimers is retarded by about 200.fold by P2-glycerate.

Lindstrom and Ho (39), based on an anion-induced pert)ur- bation of the XXR spectra of IIbCO and HbOz and on the known locus of PZmglycerate binding in deosyHb (40, 41), suggest that the binding site of organic polyphosphates is between the NHz termini of the p chains as in the deosygenatecl derivative. Thus it would appear that Pz-glycerate might serve as an ionic cross-linking agent which contributes to the forces holding to- gether a pair of c@ dimers. The cross-link would contribute about 560 cal per mole to the stability of liganded tetrameric hemoglobin compared to the dimeric state at 20”.

It is not easy to see how inorganic phosphate could serve in a similar cross-linking fashion, as is suggested by its ability to depress the apparent extent of dimerization (Table I). It is perhaps possible that two phosphate anions, bound between the NHlmterminal group of each p chain and the corresponding t-am- monium group of lysine p82 (as suggested by &none for deoxyHb (40)) could interact, perhaps by means of hydrogen bonding between the -0-H of 1 phosphate and an osygen atom of the second. Alternatively, the change in tertiary structure shown in the study of Lindstrom and Ho (39) might) lead also to an increased stability of the quaternary structure in the pres- ence of anions. This explanation seems unlikely. The effects of Cl- and phosphate observed in the NMR experiments are apparently very similar, whereas Cl- enhances dimerization of liganded hemoglobin (8, 30, 36), and inorganic phosphate ap- parently inhibits it’. Elucidation of the inorganic phosphate effect will require further experimentation.

4 This assumption may be an oversimplification. Caldwell and Nagel (35) indicate that P2-glycerate binding to oxyHbA cannot Acknozclledgments-I thank Professor J. F. Taylor for making

be described by a single equilibrium constant. his stopped flow spectrophotometer available for modification.

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2885

I am further indebted to Professor R. J. DcSa for generously 19. GR.\Y, R. I)., .LND GIISSON, Q. H. (1971) J. Viol. Chem. 246, 71687174 supplying the wiring diagrams, computer programs, and advice

for con&ucting the computer intcrfacc usctl in data collection. 20.

The University of Louisville Toward Greater Quality Committee 21.

supplied part of the financial support necessary to purchase the

components for the interface. 00

B.~NNISRJEE, R., ALPERT, Y., LI~TEILRIER, F., AND WILLIAMS, R. J. I’. (1969) 1~iochemistr?/ 8. 2862-2867

I-IoDGsL\N, c. 11.; ed. (1956) H&book of Chemistry and Physics, p. 1006, 38th Ed, Chemical Rubber Publishing Co., Cleve- land, Ohio

GIBSON, Q. H., AND MILNES, L. (1964) Biochem. J. 91,161-171 UxSa, R. J. (1970) Anal. Biochem. 36, 293-303 ~>ESA, 1~. J. (1972) in Computers in Chemical and Biochemical

Research (KLOI’FENSTEIN. C. D.. AND WILICINS. C. L.. eds) Vol. I, p. ‘185, Academic Press, New York ’ ’ ’

BEVJNGTON, I’. R.. (1969) Data Reduction and Error Analysis for the Physical Sciences, pp. 134-148, McGraw-Hill, New York

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Robert D. GrayLiganded Hemoglobin

The Effect of 2,3-Diphosphoglycerate on the Tetramer-Dimer Equilibrium of

1974, 249:2879-2885.J. Biol. Chem. 

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