Kinetics of p-Mercuribenzoate Binding to Sulfhydryl Groups on the Isolated Cytoplasmic Fragment of Band 3 Protein EFFECT OF HEMOGLOBIN BINDING ON THE CONFORMATION*

Hemoglobin binds to the cytoplasmic domain of band 3 protein (CDB3) at physiologic pH and ionic strength in an oxygen-linked fashion, with deoxyhemoglobin having the higher affinity, The evidence in the litera-ture suggests functional communication between the hemoglobin-binding site on CDB3 and the anion transport sites within the membrane-bound domain of band 3. Since t4e hemoglobin-binding site is estimated to be over 200 A from the transport domain, the functional communication hypothesis would require the existence of long-range, global changes in the CDB3 dimeric quaternary structure consequent to hemoglobin bind- ing. In this report sulfhydryl reactivity towardp-mer-curibenzoate is studied in an attempt to identify such long-range conformational changes. Formation of stoi- chiometric hemoglobin/CDB3 complexes is shown to produce major changes in sulfhydryl reactivity. Since the sulfhydryl pocket of CDe3 is known to lie at the dimeric interface over 100 A from the hemoglobin- binding site, the observed changes in reactivity suggest that hemoglobin complexation induces a global change in quaternary structure of the CDB3 dimer. This change offers a mechanism to explain functional connections between CDB3-binding sites and the anion transport sites on band 3. The existence of such long-range

over 200 A from the transport domain, the functional communication hypothesis would require the existence of long-range, global changes in the CDB3 dimeric quaternary structure consequent to hemoglobin binding. In this report sulfhydryl reactivity towardp-mercuribenzoate is studied in an attempt to identify such long-range conformational changes. Formation of stoichiometric hemoglobin/CDB3 complexes is shown to produce major changes in sulfhydryl reactivity. Since the sulfhydryl pocket of CDe3 is known to lie at the dimeric interface over 100 A from the hemoglobinbinding site, the observed changes in reactivity suggest that hemoglobin complexation induces a global change in quaternary structure of the CDB3 dimer. This change offers a mechanism to explain functional connections between CDB3-binding sites and the anion transport sites on band 3. The existence of such longrange conformational changes would imply that the CDB3 dimer is poised to function as a cytosolic arm or lever in order to modulate the global structure of the porter.
Band 3 is a multifunctional transmembrane protein found in the red blood cell (1,2). The integral domain functions to exchange anions while a 45-kDa cytosolic extension (known as CDB3)' offers binding sites for cytoskeletal proteins (3)(4)(5)(6), hemoglobin (7, 8), and certain glycolytic enzymes (9, 10 for reviews). Oxygen-linked hemoglobin binding to CDB3 (11- 13) has been observed under physiological conditions of pH and ionic strength (13), while hemoglobin binding has been shown to increase the rate of anion exchange (14). Another interaction at the hemoglobin site involves hemichrome binding which appears to somehow disrupt the naturally occurring band 3 connections with the cytoskeleton (10). In order to rationalize such linkages between band 3 subdomains, a conformational change in CDB3 of global consequence needs to be postulated.
Early studies seeking conformational connections between CDB3 and the anion transport domain failed to demonstrate linkages (10,15). However, newer evidence supports such connections. Stoichiometric coverage of the band 3 transport site with 4,4'-diisothiocyanostilbene-2,2'-disulfonate lowers hemoglobin affinity for CDB3 sites (8,14,16). Stilbene disulfonate binding to band 3 diminishes spectrin and ankyrin extractability (17) and specifically changes the conformation of CDB3 within the intact molecule (18). A new mutant form of band 3 shows altered connections between the ankyrinbinding site on CDB3 and the anion transport sites such that the transport rate is higher and the ankyrin binding capacity is lower compared to normal (19). Removal of CDB3 using genetic engineering technology causes a diminution of anion transport (20). Phosphorylation of CDB3 significantly enhances anion exchange (21). Hemoglobin binding to CDBB causes several significant changes in the kinetic parameters involved in reversible stilbene disulfonate binding (22). Cellular deoxygenation to 50% saturation reduces macromolecule binding to exofacial band 3 sites involved in cellular aggregation (23). Finally, Racker and his co-workers (14) have very recently shown that hemoglobin potentiates phosphate transport in a reconstituted band 3-phospholipid system. With this evidence favoring functional connections between various band 3 sites, what is the structural basis for communication?
This report presents results identifying hemoglobin-induced changes in CDBB quaternary structure which can explain how connections between otherwise distant sites may occur. The effect of stoichiometric addition of hemoglobin on the PMB reactivity of CDB3 sulfhydryl groups is studied. The use of PMB reaction kinetics to detect protein conformational changes is well established (24, 25). Its usefulness in the present setting lies in the fact that the CDB3 dimer is a highly elongated structure (about 250 A in length (10)). The sulfhydryl groups are clustered in the COOH-terminal half of the monomer, perhaps over 100 A from the acidic NH2terminal hemoglobin-binding site (lo), while there are no sulfhydryl groups in the NHz-terminal 23-kDa half of the fragment where hemoglobin binds.

MATERIALS AND METHODS
The chemicals and enzymes used in this study came from Sigma. Recently out-dated human red cells were obtained from the Omaha Pharmacia LKB Biotechnology Inc.
Chapter of the American Red Cross. Column materials were from The basic methods for the preparation of isolated CDBB have been described (8,26,27) and generally follow the procedures given by Bennett and Stenbuck (28) and Appell and Low (29). CDB3 concentration was determined by its optical density at 280 nm using the previously established extinction coefficient at 280 nm of 27 mM" cm" (26).
Purified hemoglobin was prepared by standard methods in this laboratory (30) and converted to the CO form. HbCO was dialyzed in 200 mM phosphate, pH 7.15, and reacted with NEM in the same buffer for 1 h (final concentrations were 1 mM heme and 3 mM NEM). This was followed by extensive dialysis in 5P6. The reaction was considered to be complete when addition of PMB showed no change in absorbance at 255 nm.
Stock solutions of 100 and 200 FM PMB solutions were prepared in 5P6 as follows. A 10 mM PMB stock was prepared in 0.1 N NaOH. An appropriate amount of this was added to 5 mM monobasic phosphate so that the above concentration would be obtained when brought to volume. Before being brought to volume, the solutions were adjusted to pH 6.0 with 5 mM monobasic phosphate to which a few drops of HCI were added (about 2 drops of 1 M HCl in 150 ml of 5P (monobase)). Once adjusted to pH 6, the PMB stocks were used to make volumetric dilutions in 5P6 for the kinetic studies. One problem which can occur in working with high concentrations of PMB is the development of a whitish, colloidal-like solution in acidic conditions. Since reactions were to be studied as a function of PMB concentration, it was necessary to ensure that no precipitate developed. This was always checked through both visual and spectrophotometric inspection and was found not to be a problem over the concentration range studied.
The kinetic studies were performed in a Gibson-Durrum stoppedflow apparatus equipped with a Xenon lamp and interfaced to a Northstar computer with software provided by On-line-Instrument-System (OLIS, Athens, GA). Stopped-flow data collection was standardized as follows. All reactions were measured at 255 nm in a 2-cm path length cuvette. Slits were set at 0.4 mm. The drive syringes and the reaction cell were in a regulated water bath at 25 "2. Any given reaction at each PMB concentration was the sum of five reactions with each containing 300 data points.
Curve fitting was accomplished using a program capable of performing weighted nonlinear fits (31).
Static titrations of CDB3 were performed in a Hitachi spectrophotometer.

RESULTS
Numbers of Reactive Sulfhydryl Groups on CDB3"Five reactive sulfhydryl groups exist on band 3 (32-34). TWO are located on the CH35 (the 35-kDa integral subdomain of band 3 generated by extracellular chymotrypsin digestion) integral subdomain while three are located on CH65 (the 65-kDa fragment of band 3 generated by extracellular chymotrypsin digestion) (Fig. 1). A static titration of the CDB3 sulfhydryls is shown in Fig. 2. There was 10 p~ of band 3 monomer present. The saturation point was found to be 18 f 2 pM. The results indicate the presence of 2 mol of sulfhydryl groups/ mol of CDB3, in agreement with one estimate of Rao and Reithmeier (33) sulfhydryl (C,). The latter site is near an internal trypsin cleavage site (Ti). These sites define two major domains of band 3, the integral, 55-kDa domain and the 45-kDa, water soluble, cytoplasmic domain (CDB3). The latter contains the hemoglobin-binding subdomain at the extreme NHz terminus. There is a second trypsin site which leads to cleavage of CDBB into two subdomains. The NHz-terminal 23-kDa subdomain contains the hemoglobin-binding site but is devoid of reactive sulfhydryl groups while the COOH-terminal subdomain contains the two reactive sulfhydryls studied here.  Fig. 5 gives the formula for the dependence of kobe on PMB concentration based on the mechanism. Equation 2 was used to fit the data shown in Fig. 4. The lines come from the fits which satisfactorily represent the data. The values of K-l and k+* for each phase are given in Table I. The fast phase has a 4-fold higher affinity and 16-fold larger intrinsic rate constant than the slow phase.
Kinetics of PMB Binding to Stoichiometric CDB3INEM-HbCO Complexes-The difference in PMB reactivity between CDB3 and the hemoglobin-bound complex is shown in Fig. 6. Both PMB reaction rates of the complex slowed. Graphical comparison of apparent rates in double reciprocal form (Fig.  7) shows that hyperbolic kinetics are followed and that the complex has a considerably lower PMB affinity at each sulfhydryl. The fast-phase sulfhydryl of the complex has a 6-fold lower PMB affinity while the affinity of the slow phase is lowered 9-fold (Table I). The change in corresponds to a 1.1 kcal/mol difference in free energy for the fast phase and a 1.3 kcal/mol difference for the slow phase. These changes represent the predominant reason for the slowing of apparent  Table I. rates since maximal intrinsic chemical reaction rate constants increase 2-fold.

DISCUSSION
Physical-chemical and hydrodynamic studies indicate an elongated CDB3 dimeric structure of perhaps 250 A (10). The 23 amino acid, acidic NH2-terminus of each monomer has been shown to constitute the exclusive hemoglobin-binding site ( Fig. 1) (35). The NHZ-terminal 23-kDa piece which contains that site is devoid of sulfhydryl groups, while the COOH-terminal 22-kDa piece contains the two reactive groups examined here (Fig. 1) (36, 37). Although the two sulfhydryl groups on one monomer are separated by about 15-kDa of amino acid residues (36,37), each group can participate  Table I).

TABLE I Kinetic constants for the reaction of PMB with CDB3
The constants were determined from weighted, nonlinear least squares fits of kb. versus PMB concentration according to Equation  Table I. The conditions were as in Fig. 3. containing the cysteine cluster (10). The kinetic results of this paper are consistent with an interfacial "pocket of sulfhydryls" hmothesis. PMB would first me-eauilibrate with the cally with either sulfhydryl. This physical picture would be expected to reflect a mechanism like that in Fig. 5. If PMB can bind to each group without steric hindrance, then the pocket must be large enough to accommodate two PMB molecules. The inherent reactivity of each group would then reflect microstructural differences within the pocket.
The large reduction in initial PMB affinity with hemoglobin complexation (Fig. 7 and Table I) would indicate a partial occlusion of the interfacial pocket of sulfhydryls according to the physical picture just discussed. This proposal is illustrated schematically in Fig. 8. The four sulfhydryls are shown to be more readily accessible to PMB in the absence of hemoglobin. Formation of the complex is shown to change the quaternary structure, thereby occluding the entrance to the sulfhydryl pocket at the interface. In this model hemoglobin would not physically block the sulfhydryls. Although it is not possible to distinguish between direct steric and allosteric competition, the allosteric hypothesis is favored since the available evidence strongly suggests that CDB3 has a very elongated structure (10). The inability to cross-link hemoglobin and CDB3 sulfhydryls when the two form a complex in solution (27), may support the allosteric linkage hypothesis.
The view that an interfacial sulfhydryl pocket becomes occluded to a small molecule like PMB consequent to changes in quaternary structure may be supported by a thermodynamic comparison to a well-known example of interfacial pocket closure to a small molecule. The example chosen for this comparison is the difference in the binding of 2,3-diphosphoglycerate to oxy versus deoxyhemoglobin. The binding site between the / 3 chains in oxyhemoglobin is occluded to organic phosphates while in deoxyhemoglobin the pocket is open (39).
The various binding data have been reviewed by Bunn and Forget (40). Binding to oxyhemoglobin occurs with a K d of 3 halves of the monomer are separated by the so-called "proline hinge" (10). The COOH-terminal half of each monomer contains the two reactive sulfhydryl groups while the NHz-terminal half contains the acidic last 23 amino acids which serve as the exclusive hemoglobinbinding site (35). Hemoglobin binding to the NH1 terminus is proposed to cause a change in quaternary structure of the CDB3 dimer such that the pocket of sulfhydryls at the interface between monomers is less accessible to PMB. This would explain the substantial lowering protein ai the entrance to the pocket befoie reacting chemiof PMB initial affinity upon hemoglobfn binding. One perdurable question in this field concerns the absence or presence of a linkage between CDB3 sites and the anion transport sites on band 3. If the band 3 transport mechanism involves a structurally rigid protein channel, there would be little reason to look for hemoglobin modulation of anion exchange. On the other hand, if each band 3 monomer contained an anion transport domain whose function was sensitive to global conformational changes, then modulation of anion exchange by hemoglobin binding would become a more likely possibility. Work with covalent 4,4'-diisothiocyanostilbene-Z,2'-disulfonate binding has shown linear activity-labeling correlation plots indicating an absence of intersubunit interactions (1, 2). Yet, linear inhibition does not preclude interprotomer allosterism if 4,4'-diisothiocyanostilbene-2,2'disulfonate binding were to uncouple naturally occurring interactions.
Direct evidence for "half-of-the-sites" inhibition by pyridoxal 5'-phosphate (a substrate and affinity probe) has been recently published by Salhany and co-workers (41). Evidence was also presented that reversible 4,4'-dinitrostilbene-2,2'disulfonate binding eliminated the half-of-the-sites pattern, yielding linear correlation plots. This shows that stilbene disulfonates can uncouple the site-site interactions for a transported anion. Since transport kinetics for physiologic anions show allosteric patterns in the form of partial substrate inhibition (1, 2), allosteric coupling between band 3 protomers seems probable. Evidence that allosteric coupling may explain the partial substrate inhibition effect, comes from the fact that this type of inhibition can be relieved by addition of an external cross-linking agent to band 3 (42). Since the substrate inhibition effect is dominated by an intracellular chloride site (43), the transformation to hyperbolic kinetic patterns resulting from the addition of an extracellular agent offers strong support for the view that anion exchange is indeed modulated by allosteric events.
If interprotomeric allosterism exists for the band 3 porter, then hemoglobin may serve as a heterotropic allosteric affector of the transport activity through modulation of quaternary structure. This modulation, since it would be linked to the state of hemoglobin oxygenation (13), could provide a direct connection between the oxygen transport and the C02 transport systems of the red cell. The asymmetric structural disposition of band 3 in the membrane speaks of such a direct connection. Only about 10% of the copies of band 3 are directly bound by red cell ankyrin (3). The remaining copies of the porter may form a looser association, so explaining the surprisingly rapid exchange of ankyrin-free for ankyrin-bound band 3 (44). Dynamic band 3 exchange has the potential to be modulated by the state of cellular oxygenation if deoxyhemoglobin binds to ankyrin-free band 3 and stabilizes that quaternary structure of CDB3.
Linkage between the hemoglobin-binding site and the anion transport sites may be needed during extreme exercise, when bicarbonate transport through band 3 is thought to become rate limiting to COS transport (45). Increased steady state levels of deoxyhemoglobin during extreme exercise could favor greater fractional hemoglobin binding to band 3 (13). The attendant increase in the rate of anion exchange (14) afforded by increased binding could relieve the putative rate limitation imposed by an otherwise immutable anion exchange rate (45). Even as small a change in rate as a factor of two could make a significant difference in CO, transport during exercise, owing to the steepness of the curve relating anion exchange to COS efflux at the lung (45).
In summary, the hemoglobin-induced conformational change in CDB3 identified here offers a basis upon which to explain an otherwise vague functional connection between the NH2-terminal domain of band 3 and the COOH-terminal domain containing the transport sites. The physiological significance of this mechanism for site communication on band 3 may become more apparent as band 3s emerging function as an allosterically modulated porter (41) becomes better defined.