THE JOURNAL OF BIOLOGICAL. CHEMISTRY Vol. 254, No. 23, Issue of December 10, pp. 12204-12208, 1979 Printed in U.S. A.

X-ray Diffraction and Solution Studies of Specifically Carbamylated Human Hemoglobin A EVIDENCE FOR THE LOCATION OF A PROTON- AND OXYGEN-LINKED CHLORIDE BINDING SITE AT VALINE la*

(Received for publication, April 3, 1979)

Stephen O’Donnell, Rosalie Mandaro, and Todd M. Schuster

From the Biochemistry and Biophysics Section, Biological Sciences Group, University of Connecticut, Storrs, Connecticut 06268

Arthur Amone.+

From the Department of Biochemistry, The University of Zowa, Iowa City, Zowa 52242

Previous studies have shown that about 30% of the alkaline Bohr effect in human hemoglobin can be at- tributed to the NHz-terminal amino group of Val la (Kilmartin, J. V. (1977) Trends Biochem. Sci. 2, 247- 249). Rollema et al. ((1975) J. Biol. Chem. 250, 1333- 1339) have shown that about the same percentage of the alkaline Bohr effect is due to the ability of chloride to bind preferentially to deoxyhemoglobin. In this pa- per we present the results of x-ray diffraction studies on human deoxyhemoglobin carbamylated at Val la, together with solution studies on the chloride depend- ence of the alkaline Bohr effect and oxygen affinity of specifically carbamylated human hemoglobin. The x- ray studies reveal the presence of two inorganic anion binding sites associated with Val la and show that these sites are blocked when Val la is carbamylated. Specifically, one site is located between the a-amino group of Val la and the /3-hydroxyl group of Ser 131a on the same a chain, and the other site is positioned between the a-amino group of Val la and the guanidin- ium group of Arg 141a on the opposite a chain. The solution experiments show that almost all of the chlo- ride-induced alkaline Bohr effect is eliminated when Val la is carbamylated, and that the difference in oxy- gen affinity between unmodified and Val la-carba- mylated hemoglobin is greatly reduced at chloride con- centrations below 10 mM. Together, these studies imply that chloride binding to one or both of the above sites is strongly linked to the oxygen and proton affinity of hemoglobin.

Heterotropic allosteric interaction in hemoglobin involving C02, protons, and organic phosphates occur through binding at the NHz-terminal amino groups. It is now well documented that the oxygen-linked binding of CO2 to hemoglobin is due to carbamino formation at the NH2 termini of all four subunits

* This research was supported by National Institutes of Health Grant AM-17563 and National Science Foundation Grant GB-43803 to A.A., by National Institutes of Health Grant HL-17494, National Science Foundation Grant PCM-20041 to T.M.S., and by National Institutes of Health Fellowship HL-05222 to S.O. The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of National Institutes of Health Research Career De- velopment Award AM-00099.

of the a& tetramer,’ that the NH2 termini of the a chains contribute a large fraction of the alkaline Bohr effect, and that the NH2 termini of the ,8 chains form part of the organic phosphate binding site (1, 2). Our preliminary reports (3, 4), as well as the studies of Chiancone et al. (5), Nigen et al. (6), and Arnone and Williams (7) indicate that the NHz-terminal amino groups of the a chains may also be sites for the oxygen- linked binding of inorganic anions.

Functional studies of the NH2 termini of hemoglobin were greatly facilitated by the discovery of Kilmartin and Rossi- Bernardi (8, 9) that cyanate can be used to carbamylate selectively the a-amino groups of the a or the /3 chains, or both, to produce the derivatives azC/32, a&‘, and (YZ’~Z’. Sub- sequently, Williams et al. (10) found that gram quantities of these derivatives could be prepared by an efficient chromat- ographic method that takes advantage of the change in net charge that results from the stronger binding of organic phos- phates to az”j?z. Since these derivatives were first used by Kilmartin and Rossi-Bernardi (8, 9) to study the oxygen- linked interactions of CO2 and protons with hemoglobin, they have been the subject of many other studies, and as a result many detailed properties of carbamylated hemoglobins are now known. In particular, the studies on carbamylated he- moglobin S show cyanate to be a potent antisickling agent (11, 12).

In this paper we report the results of x-ray diffraction studies on the deoxy structure of azCP2 together with solution studies on the chloride dependence of the alkaline Bohr effect and oxygen affinity of specifically carbamylated hemoglobins. We find that inorganic anions normally bound to two sites at the NH2 termini of deoxyhemoglobin are lost as a result of carbamylation. Moreover, the binding of chloride ions to one or both of these sites is linked to the oxygen and proton affinity of hemoglobin.

MATERIALS AND METHODS

Protein Preparation-Blood was obtained from a normal adult donor, with EDTA as the anticoagulant. The main component of hemoglobin, HbAo, was isolated as oxyhemoglobin using the method of Williams and Tsay (13). Hemoglobins specifically carbamylated at

’ The abbreviations used are: a&, (YZ’/&, LX&‘, and azCPzC, the unmodified hemoglobin tetramer and hemoglobin specifically modi- fied at the NH2 termini of the (Y chains, /3 chains, and all four subunits, respectively; bis-Tris, bis(2-hydroxyethyl)imino-tris(hydroxymethyl)- methane; EDTA, ethylenediaminetetraacetic acid; DPG, 2,3-diphos- phoglycerate; IHP, inositol hexaphosphate.

12204

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Hemoglobin Carbamylated at Val la 12205

the NH*-terminal valines of each subunit (uzr/32, ol&‘, al’P2’) were prepared in the oxygenated form using the procedure of Williams et al. (IO). Since the specifically carbamylated derivatives are separated on a carboxymethylcellulose (CM-52, Whatman) column using phos- phate buffer containing 10m4 M IHP (sodium salt, Sigma), they had to be stripped subsequently of IHP. This was achieved by passing 10 ml of each derivative (1.8% in heme) through a drained mixed bed resin column (29 x 45 mm) (Bio-Rad AG 501-X8 (D)) with a thin layer of the H’ form resin (Bio-Rad 5OW-X8) placed at the bottom of the column to remove any residual amines. The column was previously equilibrated with Cog-free water at 4°C. The hemoglobin was eluted with a small volume of Con-free double distilled water into 0.05 M

Tris (enzyme and buffer grade, Schwarz-Mann) buffer, pH 7.5. Sam- ples were pressure concentrated at 4°C (Amicon Ultrafiltration Cell, DM-5 membrane, Amicon Corp., Lexington, Mass.) and frozen rapidly in liquid nitrogen.

X-Ray Diffraction Studies--Single crystals of purified ollcpl and native human hemoglobin were grown in the deoxy state from solu- tions of concentrated ammonium sulfate according to the method described by Perutz for deoxyhemoglobin A (14). All manipulations of the crystals were carried out in a glove box filed with high purity nitrogen.

Diffraction data were collected to a resolution of 3.5 A on an Enraf- Nonius CAD4 diffractometer using the w scan mode. One crystal was used for each set of data (15,ooO reflections). An empirical correction for radiation damage was incorporated into the crystal scaling calcu- lations as described previously (15). This correction factor never exceeded 10%. Corrections for x-ray absorption were calculated and applied according to the method of North et al. (16).

Difference electron density maps were calculated using the known phases of deoxyhemoglobin A (15) and the difference amplitudes (I Fl”,“&d Hb - lFIn&, then symmetry averaged about the molecular 2-fold rotation axis that relates the 2 equivalent up dimers (17).

Proton Titrations-Protein solutions were dialyzed at 4°C as ox- yhemoglobin against salt solutions (0.10 M KC1 or 0.0050 M KCl) for at least 4 days. One day prior to the end of the dialysis the oxyhe- moglobin was titrated at 20°C to pH 7.2 in a CO*-free atmosphere. At the completion of the dialysis, the protein solutions were diluted with their respective salt solution to a concentration of approximately 400 pM in heme, using the value of 14.7 mM-’ cm-’ as the extinction coefficient at 576 nm for oxyhemoglobin at 21.5”C, and then trans- ferred to a 5°C water-jacketed tonometer. The samples were deoxy- genated by passing ultrapure N, (Airco Grade 5) over the samples while the thermostated tonometer was gently shaken. The samples were deoxygenated in this manner for at least 1 full day. Visible absorption spectra showed that deoxygenation of the highest oxygen affinity hemoglobin samples was complete within this time. All titra- tions were performed at 20°C.

The titration cell was kept COz- and Os-free by passing the N* outflow from the tonometer into the water in the cell. The tip of a combination electrode (Radiometer model GK 2351C), previously standardized, was kept immersed in water in the cell immediately prior to each titration to prevent drying. At the start of each titration the water in the cell was removed using a syringe and approximately 4 ml of the hemoglobin solution was anaerobically transferred from the tonometer directly into the 20°C cell. Since Rollema et al. (18) have shown that the magnitude of the chloride dependence of the alkaline Bohr effect is constant over the pH range 7.4 to 7.6, no effort was made to obtain greater starting precision than this. However, if the starting pH of the deoxygenated hemoglobin was above 7.65 the sample was discarded. The pH of the fully deoxygenated solution was recorded and the hemoglobin was then liganded with CO (A&o, Grade 2.3) by drawing some of the solution into a CO-ftied syringe, mixed, and returned to the cell. This procedure was repeated several times to insure complete formation of HbCO. The solution was then back-titrated with standardized NaOH using a micrometer syringe (Alga). Conditions were chosen so that the overall volume change was less than 0.5%. Conductivity measurements on a 5.0 mM KC1 solution showed that the change in chloride concentration due to electrode leakage was negligible over the time course of a titration. An absorp- tion spectrum of each sample was obtained at the end of each titration to check for saturation with CO and for methemoglobin formation. Samples containing more than 5% methemoglobin were discarded.

Oxygen-binding Isotherms-Oxygen equilibrium measurements at all but the two lowest salt concentrations were performed at 10.0 (* 0.1) “C using a modified version of the automatic recording system of Imai et al. (19). All buffers were 0.10 M bis-Tris, with 10e4 M EDTA, prepared by titrating the free base form of bis-Tris with concentrated

HCl then adding enough NaCl to bring the buffer to the desired anion concentration. Concentrated hemoglobin solutions were dialyzed overnight against a 100-fold excess of appropriate buffer (two to three changes of dialysate), diluted to 50 to 60 pM in heme and filtered through a 5-pm membrane (Millipore). Samples were deoxygenated by passing ultrapure nitrogen (hydrated with the respective buffer using double gas dispersion tubes) into the Imai-type 2-cm optical/ polarographic cell. Oxygen saturation was monitored optically at 560 nm in a Cary 118 C spectrophotometer. Oxygen pressure was meas- ured using a polarographic oxygen sensor (Beckman model 39065). The electrode output was amplified by a high gain (approximately 10”) current to voltage transresistor, using a very stable operational amplifier (Analog Devices, model 52K). The output of the amplifier was recorded concurrently with optical measurements on a microvolt sensitivity chart recorder (Radiometer REC-61 Servograph). The electrode was calibrated before each experiment with known gas mixtures (Airco); electrode linearity in the working range (0.2 to 7.0 mm Hg) was ascertained in a similar manner. Optical spectra (650 to 520 nm) were recorded periodically during deoxygenation as a check for methemoglobin formation; samples with more than 5% methe- moglobin were discarded.

For the oxygen-binding studies at the two lowest salt concentra- tions, a 0.010 M bis-Tris buffer, pH 7.36 at lO.O”C, 0.0020 M in Cl-, and lo-” M in EDTA was used. A lower pH was used for these low salt measurements in order to try to maximize any chloride binding to deoxyhemoglobin since Garner et al. (20) have reported a pK, of 7.8 at 26°C for the Val lay of deoxyhemoglobin. The oxygen-binding studies at the lowest salt concentrations were done using an anaerobic spectrophotometer cell. The cell consists of a 1.00~cm pathlength quartz optical cell fused to a small tonometer. Tipping the cell through 90” results in the solution in the cell flowing into the tonometer where it can be rapidly equilibrated with various gases while being gently shaken in a water bath. Hemoglobin concentrations were approxi- mately 50 pM heme. For each experiment, the solutions were deoxy- genated overnight in a separate water-jacketed tonometer at 5-10°C with ultrapure nitrogen. The spectrophotometer cells were flushed with Nz for at least 3 to 4 h before each experiment. At the start of each run, the tonometer was tipped and 3 to 4 ml of the solution transferred anaerobically into the cell with a small positive pressure of nitrogen. The cell was then equilibrated in a water bath at 10.0 f 0.02”C for 10 min, placed in a specially constructed 10.0 + 0.02”C water-jacketed cell holder in the spectrophotometer and the spectrum recorded from 650 to 520 nm. After the spectrum was recorded, the cell was returned to the water bath and attached to a precision gas- mixing pump (Godard Mixing Pump, Instrumentation Assoc., Inc.) and a hydrator filled with the appropriate buffer. The gas-mixing pump is capable of producing 12 mixing ratios from 0.01 to 0.10 to an accuracy of about 0.25% of the ratio. For these experiments various percentages of a 1.04% 02/N2 gas mixture (Airco) were mixed with ultrapure nitrogen. Under these conditions the po, can be varied from approximately 0.08 to 0.8 mm Hg in 0.08 mm Hg steps. A spectrum of the fully oxygenated sample was obtained at the end of each run by allowing air to mix with the sample. Two cells were normally run in tandem, one containing u& and the other containing a,‘/?z, both in the same buffer. Duplicate samples were run for each salt concentra- tion. No methemoglobin formation was observed during any of these measurements.

Oxygen saturations ( P) were calculated from the equation

p = ( Amdoxy) - Am(t))

( Adoxy) - Addeoxy))

where A,&oxy) = absorbance at 560 nm for the completely oxygen- ated sample; Ar&deoxy) = absorbance at 560 nm for the completely deoxygenated sample; A& t) = absorbance at 560 nm for the partially deoxygenated sample at time t, corresponding to PO,(t) from the oxygen electrode or to PO, from the known gas mixtures in the tonometer method.

All po, values were corrected for the 10°C vapor pressure of water at the laboratory-measured barometric pressure.

RESULTS

X-Ray Studies

There are only three significant features in the deoxy az’/3z difference map (Fig. l), a positive difference peak and two adjacent negative peaks. The positive peak is contiguous with the native electron density of the (Y chain NHz-terminal amino

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Hemoglobin Carbamylated at Val la

FIG. 1. Fourier maps showing contiguous electron density sections which include al and (~2 chain residues Val la, Arg 14111, and Lys 127q the A helices, H helices, and heme groups of the (Y chains, as well as small parts of the a& and cy& interfaces. The features labeled X2- and X3- are inorganic anions bound to the amino group of Val In; XI is probably a water molecule (see text). All features occur in pairs related by the molecular P-fold axis of symmetry (only one member of each pair has been labeled), and the maps have been averaged about this axis. The symmetry axis is perpendicular to the plane of the paper and passes through the center of each figure. A, composite of nine two-dimensional sections (spaced 1 8, apart) of the native human deoxyhemoglobin electron density map at 2.5 A resolution (sections +23 to 15 A above the center of the tetramer). The contours are drawn at intervals of 0.15 e k” above the zero level with contours on upper sections obscuring the

lower ones. B, difference electron density at 3.5 8, resolution (sections +I6 to +18 A) of (YZ’,!?, ( white contours) superimposed on the native protein electron density ( black contours). Positive difference electron density is marked by solid white contours and upper case letters, negative difference electron density is marked by broken white con- tours and lower case letters. The contours for the difference map are drawn at intervals of kO.016 e A-“. The positive difference electron density resulting from the added carbamyl group has been labeled as Peak A. The extended negative Peak b is due the displacement of features XI and X2-. XI is probably a water molecule; XS- has been shown to be an inorganic anion binding site (see text). C, native human deoxyhemoglobin electron density (sections +15 to +lO A) with contours drawn as in Fig. 1A. D, difference electron density (sections +12 and +13 A) of a~‘/& contoured as in Fig. 1B. Negative Peak c is due to the displacement of inorganic anion X3-.

group and marks the position of the carbamyl group. One of the negative peaks is located between the a-amino group of Val la, and the guanidinium ion of Arg 141q while the other is positioned between the (Y amino group of Val la, and the (Y hydroxyl group of Ser 131a1. Each of these negative peaks superimposes on very weak features in the native electron density map (labeled X1, X2-, and X3- in Fig. l), and therefore could represent the displacement of bound water molecules or inorganic anions (sulfate or phosphate) which are present at less than full occupancy, are disordered in the crystals of native deoxyhemoglobin A, or both. We feel that XZ- and X8- represent anions since the distances of the negative peaks from the interacting protein groups (4.2 to 4.6 A) are consistent with the distances expected for sulfate (or phosphate)-protein interactions (21). This was confirmed by recent x-ray diffrac-

tion studies which showed that selenate ions bind at the positions of Xz- and XB- (but not at X1) in deoxyhemoglobin (7). X1 is probably a water molecule.

The absence of any other significant features confirms that only the (Y chain NHt-terminal amino groups have been mod- ified, and shows that carbamylation does not perturb the tertiary structure of the a: subunits.

Solution Studies

Bohr Group Titrations-The results of titrations of the alkaline Bohr groups on unmodified and specifically carba- mylated hemoglobins are presented in Table I. Comparing the a& titration with that of (YZ’~~ shows that carbamylation of the (Y chains results in a 32% reduction in the number of protons released upon ligation in 0.10 M KCl. A very similar

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Hemoglobin Carbamylated at Val la I2207

reduction is found between the a&’ and (Y~‘Pz’ titrations, indicating that the NH*-terminal amino groups on the p chains do not contribute significantly to the alkaline Bohr effect. This conclusion is verified by the fact that the titrations of a& and a&’ are identical in value. These results agree with those of Kilmartin et al. (22) who prepared the specifi- cally carbamylated hemoglobins by recombination of sepa- rated subunits. At a salt concentration of 0.0050 M KC1 smaller differences are observed in the number of protons released upon liganding a.&, ozrP2, and (~~‘/3~‘. The value for normal hemoglobin, 0.36 H’ per heme, represents a 28% reduction of the value measured in 0.10 M KCl, whereas the magnitude of the alkaline Bohr effect for ax’/?2 is, within the accuracy of the measurement, the same at both chloride concentrations. The small chloride dependent decrease in the Bohr effect for (YZ~/%~ is probably significant. However, it is clear that carbamylation of Val l/l has little or no influence on the Bohr effect at 0.10 M or 0.005 M chloride, and that almost all of the chloride- linked Bohr effect is lost when Val la is carbamylated.

Chloride Dependence of the Oxygen Affinity of a& and anCbs-In Fig. 2 we have plotted the log (~50) of (~$2 and a~‘p~ uersus the log (Cl-) at 10°C. Above 10 mM chloride (at pH 7.60) the curves are essentially parallel. However, whereas the oxygen affinity of (Y& continues to increase when the chloride concentration is decreased from 10 to 2 mM (at pH 7.36), the pSo of (Ye’& is essentially unchanged at these two chloride concentrations. This suggests that a binding site (or sites) with relatively high affinity for chloride is eliminated as a result of carbamylating Val la. Recently, Manning et al. (23) have also reported that the difference between the pm of a&

TABLE I

Summary of Bdhr effect proton titration data (number of H+/heme, AH+, released on binding CO)

Experiments were performed with 300 to 400 PM heme at 20°C and within the pH range of 7.4 to 7.6. Each entry is the average of from two to five titrations.

AH+ A(AH+)

0.10 M KC1 0.0050 M KC1

0.50 0.36 0.14 0.34 0.33 0.01 0.50 0.36 0.31 0.05

0.6 A

0.5 _

04_

03-

0.2

0.1 -

log(p5o)o.o~

- O.l-

-0.2_

-0.3_ -0.4- *

-0.5.

-0.6 f I

E

I

I 4 I

FIG. 2. Chloride dependence of the oxygen affinity of a& (j) and azc& (3) at 10°C. The data in B were collected at pH 7.60 in the presence of IO-“ M EDTA; the data in A were collected at pH 7.36 in lo-” M EDTA. Each point is the average of two measurements which disagreed by the amount shown by the error bars.

and the pSo of azcj3z is reduced at low chloride concentrations (<lo mM).

DISCUSSION

A number of studies have shown that the o-amino group of Val la is responsible for a large fraction of the alkaline Bohr effect (8,20,24). In a recent review, Kilmartin (25) concluded that 30% is the best current estimate of the portion of the alkaline Bohr effect generated by Val la. In terms of pK values, Garner et al. (20) have reported that the pK of Val la varies from 7.0 in carbonmonoxyhemoglobin to 7.8 in deoxy- hemoglobin.

At 3.5 A resolution, an electron density map of native human deoxyhemoglobin indicated the possibility of a salt bridge between the NH2-terminal amino group of each a subunit and the COOH-terminal carboxyl group of the oppo- site (Y subunit: i.e. between Val la, and Arg 141~ and between the dyad-related pair Val la2 and Arg 141ar (26, 27). Since these salt bridges do not exist in methemoglobin (28) or carbonmonoxyhemoglobin (29), they would provide a stereo- chemical explanation for that portion of the alkaline Bohr effect which is associated with the a-amino group of Val la. However, when the resolution of the human deoxyhemoglobin electron density map was extended to 2.5 A (15), Fermi (30) refined an atomic model into this map and found that the terminal carboxyl and amino groups on opposite (Y chains are too far apart (5.3 A) to interact strongly. As Fermi points out though, the high resolution crystallographic data were col- lected on crystals grown in a mother liquor of 2.3 M ammonium sulfate and 0.3 M ammonium phosphate, and therefore the possibility still exists of a salt bridge between the a-amino group of Val lcvl and the a-carboxyl group of Arg 141~~ under the low salt conditions found in the red cell.

Our difference electron density map of deoxy ozcj32 uersus deoxy ~$2 suggests another explanation for the Bohr effect associated with Val la. Along with the positive difference density showing the expected addition of a carbamyl group to Val la, negative difference peaks suggests that two inorganic anion binding sites are associated with each (Y chain NH2 terminus (see Fig. 3). One site (labeled X2- in Figs. 1 and 3) is

FIG. 3. Sketch showing the environment of the NH2-terminal amino group of the o2 chain in deoxyhemoglobin A. This region includes arginine 141~x1 (the COOH terminus fo the (~1 chain), and (~2 chain residues aspartate 126, lysine 127, and aspartate 6. These residues form salt bridges (indicated by the broken lines) which are essential for full allosteric action in hemoglobin (27). X2- and X3- are inorganic anion binding sites which are blocked in (YZ’/&. For clarity, the peptide backbone between the 1st and 5th residues of the (~2 chain has been deleted.

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Hemoglobin Carbamylated at Vu1 la

between the a-amino group of Val la, and the /3-hydroxyl group of Ser 131ar, and the second site (labeled X3- in Figs. 1 and 3) is between the a-amino group of Val la, and the guanidinium ion of Arg 141~~~. In the high salt crystals of deoxyhemoglobin used for our x-ray studies these sites must be occupied by sulfate or phosphate. Clearly, we could account for all or at least part of the alkaline Bohr effect associated with Val la by assuming that inorganic anions bind prefer- entially to deoxyhemoglobin at X2-, or X3-, or both.

Chloride, however, is the major inorganic anion in the red blood cell and in almost all in vitro experiments designed to measure the Bohr effect. The work of Rollema et al. (18) clearly shows that a large fraction of the alkaline Bohr effect is due to the oxygen-linked binding of chloride. They found that the magnitude of the alkaline Bohr effect is reduced by about 35% (at pH 7.4) if the chloride concentration is reduced from 100 to 5 mM. Our titration data of unmodified hemoglo- bin (Table I) are in good agreement with their measurements. However, we also find that carbamylation of Val la eliminates almost all of the chloride-induced portion of the alkaline Bohr effect. Therefore, it seems reasonable to conclude that at a chloride concentration of 100 mM, chloride anions bind pref- erentially to deoxyhemoglobin at sites X2-, or X3-, or both, that this binding is blocked when Val la is carbamylated, and that chloride binding is responsible for almost all of the alkaline Bohr effect which is associated with Val la. That is, the binding of chloride to sites X2-, or X3-, or both, causes the pK of Val la to increase to a value of 7.8 (20) in deoxyhemo- globin, whereas the more normal value of 7.0 (20) is observed in liganded hemoglobin where the binding of chloride to these sites is reduced or eliminated.

This conclusion is supported by the following results. 1) We find, as does Manning et al. (23), that the difference in oxygen affinity between a& and azc/12 decreases markedly when the chloride concentration is reduced below 10 mM. This implies that an oxygen-linked chloride binding site (or sites) is blocked as a result of the carbamylation of Val la. 2) Nigen et al. (6) have shown that chloride is a competitive inhibitor of the carbamylation of Val lcu, and that the inhibition is greater with deoxyhemoglobin than with liganded hemoglobin. 3) Difference electron density analysis carried out on high salt crystals of deoxyhemoglobin soaked in solutions containing selenate showed that selenate anions bind at sites XZ- and XB- (7). Recently, similar experiments with bromide-soaked crys- tals have shown that bromide anions also can bind to these sites.’ 4) From 35C1- NMR experiments on normal and car- boxypeptidase B-digested hemoglobin (i.e. hemoglobin modi- fied such that Arg 141a has been deleted), Chiancone et al. (5) concluded that chloride ions are bound “at or near Val lar

* A. Arnone and B. Foster, unpublished work.

- Arg 141~~~” in carbonmonoxyhemoglobin.

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S O'Donnell, R Mandaro, T M Schuster and A Arnonechloride binding site at valine 1 alpha.

hemoglobin A. Evidence for the location of a proton- and oxygen-linked X-ray diffraction and solution studies of specifically carbamylated human

1979, 254:12204-12208.J. Biol. Chem. 

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