the erythrocyte membrane (fluorescent probes)

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1 Proc. Natl. Acad. Sci. USA Vol. 77, No. 12, pp , December 1980 Biochemistry Kinetic study of the interaction of oxy- and deoxyhemoglobins with the erythrocyte membrane (fluorescent probes) N. SHAKLAI* AND V. S. SHARMAtf *Department of Chemical Pathology, The Sackler School of Medicine, Tel-Aviv University, Israel; and tdepartment of Medicine, T-006, University of California at San Diego, La Jolla, California Communicated by Helen M. Ranney, September 2, 1980 ABSTRACT Changes in fluorescence intensity of a membrane-embedded probe were used to study the kinetics of binding of oxy- and deoxyhemoglobin to erythrocyte membranes. For these studies, stopped-flow fluorimetric techniques were utilized. Both binding and dissociation of hemoglobin from membranes followed heterogeneous first-order kinetics. The rate constants for binding of oxyhemoglobin were about 10 times larger than those of deoxyhemoglobin; the dissociation rate constants of oxyhemoglobin were about one-quarter those of the unliganded form. The results are discussed in light of the steady-state binding constants previously derived for both oxyand deoxyhemoglobin. In previous studies (1-3) the steady-state characteristics of the interaction of HbO2 and Hb with erythrocyte membranes were described. In those studies, Hb was used to quench the fluorescence intensity of a membrane probe. HbO2 exhibited a higher affinity for the membrane than did Hb. However, there are certain inherent drawbacks in steady-state fluorescence studies of the binding of Hb to erythrocyte membranes. In order to avoid trivial quenching, these experiments were limited to Hb concentrations not exceeding 1,M (heme basis). At these low concentrations, HbO2 is significantly dissociated into dimers and it was therefore uncertain whether the heterogeneity observed in the binding of HbO2 was due to the heterogeneity of binding sites on the membrane or to different affinities of the dimer and tetramer for the same binding site (4). Inability to use higher Hb concentrations in steady-state studies in some systems precluded the study of the binding of Hb to low-affinity sites (2). Light scattering measurements provide an alternative approach in which higher concentrations of Hb can be used. However, at higher Hb concentrations, erythrocyte ghosts tended to agglutinate, leading to artifacts that interfered with the analysis of the binding data (3). In this communication we present the results of kinetic studies on the reactions of Hb and HbO2 with erythrocyte membrane by using a fluorescence quenching technique. This technique is free from limitations due to trivial quenching and permits the use of higher concentrations of Hb. The effect of dimer formation in the binding of Hb to erythrocyte ghosts could then be observed. The kinetic approach also made possible study of the binding of Hb to low-affinity sites on ghosts. The kinetic results are compared with the results of earlier steady-state studies of Hb-erythrocyte interactions. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact MATERIALS AND METHODS Glyceraldehyde-3-phosphate. dehydrogenase (GAPD) was purchased from Sigma; 9-(1,2-anthroyl)stearic acid (AS) was from Molecular Probes (Plano, TX). All other chemicals were reagent grade. Hb was prepared by lysis of washed erythrocytes in water and toluene, followed by passage through a Sephadex G-25 column. For complete deoxygenation, a sample of concentrated Hb was equilibrated with nitrogen and then was diluted with oxygen-free buffer. In experiments with Hb, oxygen-free solutions containing 0.3 mm dithionite were used in the two syringes of the stopped-flow apparatus containing erythrocyte ghosts or Hb. The dithionite concentration was kept low in order to avoid significant changes in the ionic strength of the buffers. At the conclusion of the kinetic runs, the reaction mixture was transferred anaerobically to a sealed cuvette and the Soret spectrum of the solution was measured to ascertain that the Hb had remained deoxygenated during the experiment. Hb-free membranes were prepared as described (1). To each 1 ml of packed ghosts (containing about 2.5 mg of lipid) 25 Mig of AS was added (1, 2). GAPD as supplied by Sigma was dialyzed against the desired buffer immediately before use; the concentrated GAPD solution was cleared of denatured insoluble material by centrifugation. The protein concentration was calculated by using e2o = 5 X 105 M-1 cm-1 (5). Kinetic measurements were performed by using a Durrum model D1 0 stopped-flow spectrophotometer operated in the fluorescence mode. The reaction mixture was excited with a xenon lamp, and stray light was removed from the exciting beam with a 2-mm Corning glass 7-54 filter. Emitted light was collected through a Corning 3-73 filter which passes light above 380 nm. Light absorption measurements were made with a Cary 14 recording spectrophotometer. An Hitachi Perkin- Elmer 44B spectrofluorimeter was used for steady-state fluorescence measurements. All kinetic experiments were made at 20 C in 5 mm phosphate buffers at ph 6.0. Kinetic studies at this ph could be compared with data obtained in earlier steady-state observations. RESULTS Extent of the Reaction. Fluorescence intensity is correlated with the density of Hb molecules on the erythrocyte membrane Abbreviations: AS, 9-(1,2-anthroyl)stearic acid; GAPD, glyceraldehyde-3-phosphate dehydrogenase. t To whom reprint requests should be addressed.

7148 Biochemistry: Shaklai and Sharma by the following steady-state expression: Io/I - 1 = Kq(T [1] in which Io is fluorescence intensity of the ghosts before binding Hb or at time zero of mixing

$2 and 3, Aht*10I ot 0%, =Atit* It. [4] Under the conditions of our experiments the fraction of fluorescence quenched was less than 10%, and therefore IX a~ot AIt I - 1 or -.$

2 7148 Biochemistry: Shaklai and Sharma by the following steady-state expression: Io/I - 1 = Kq(T [1] in which Io is fluorescence intensity of the ghosts before binding Hb or at time zero of mixing with Hb, I is fluorescence intensity after quenching due to binding of Hb, Kq is effective quenching constant, and a is bound Hb density in mol/cm2. In kinetic experiments, a and I are time dependent and therefore Eq. 1 can be written as: Io- = t = AI t =Kq at [2] it It It and at infinity Ai. = KqO%.(AI. = Io - I.). [3] From Eqs. 2 and 3, Aht*10I ot 0%, =Atit* It. [4] Under the conditions of our experiments the fraction of fluorescence quenched was less than 10%, and therefore IX a~ot AIt I - 1 or -. [5] The general rate equation for a set of n independent first-order reactions becomes: n n -k L AtI i=l = L AJae k.t i=l Because at/a. measures the extent of reaction, it was replaced by the time-dependent quenched fluorescence intensity, At/WA, as a measure of the reaction rate. Kinetic Rate Constants. The study was conducted with "open ghosts." Because the Hb binding sites are located on the [6] membrane surface (inner), we must assume that in open ghosts the free Hb concentration at the layer near the surface is the same as that of the outside bulk solution and that the system is free from the problems of unstirred layers (6). The reaction between Hb and membrane sites (Ms) may be written as: Hb + Ms 0.P. in which P is the Hb-MS complex and the apparent rate equation is d[p] _ d(a)t = kapp[hb][ms]. The dimensions of Ms and P will be mol/cm2 and the dimension of water bulk Hb will be mol/cm3. The apparent rate constant is therefore expressed in mol/cm3.sec, the usual dimensions of second-order rate constants. Blank runs were made by mixing fluorescent-probe-labeled erythrocyte with buffer to ascertain the absence of artifacts. Dissociation of Hb from the Membrane. To measure the rate of dissociation of Hb from the membrane, we made use of earlier findings that GAPD and Hb compete for binding at the same inner surface sites (2). The affinity of Hb for the membrane sites was found to be of the same order as that of GAPD (7) and lower for the low-affinity sites (8). Therefore, in studies of the dissociation of Hb from membrane sites, GAPD in 100-fold excess over Hb will readily compete for both highaffinity and low-affinity sites. Hb-MS - Proc. Natl. Acad. Sci. USA 77 (1980) Ms + Hb [la] r..-i CQ cn 0 FIG. 1. Typical stopped-flow fluorimetric kinetic oscilloscope tracing of HbO2 binding (B) and release (A) from erythrocyte membrane sites. Ghosts concentration, 1010 cells per liter; time elapsed, 10 sec in A and 1 sec in B FIG. 2. Semilogarithmic plot of kinetics of HbO2 binding to erythrocyte membranes. Membrane concentration, 5 X 109 cells per liter; Hb, 3.3 ym after mixing. Other experimental conditions as in Fig. 1. The curve is resolved into two first-order components. The dashed line extends the linear portion of the slow phase. A, Experimental data; 0, fast phase resolved.

3 Biochemistry: Shaklai and Sharma GAPD + MS Ms-GAPD - GAPD + Hb-MS 4 Ms-GAPD + Hb. [lb] [II] Proc. Natl. Acad. Sci. USA 77 (1980) 7149 By varying the concentrations of GAPD, it can be readily ascertained that in I the back reaction is insignificant. HbO2 Binding and Release from Membranes. The "on" reaction rate was found to depend in a linear fashion on the HbO2 concentration in the range AM (heme concentration). On the other hand, the reaction rate was independent of ghost concentration in the range studied, 8 X 108 to 8 X 1011 ghosts per liter. An oscilloscope tracing of a typical experimental record for the binding of Hb to erythrocyte membranes is shown in Fig. 1B. The reaction time course is shown in Fig. 1A. Fig. 2 shows the first-order kinetic plot; rate constants for the slow and fast phases were resolved by usual graphic methods. The dissociation of HbO2 bound to erythrocyte membrane was studied by displacing HbO2 from erythrocyte ghosts (8 X 108 cells per liter) saturated with Hb (0.1 mm, heme basis). The concentration of GAPD varied in the range km. The reaction rates were independent of the enzyme and ghost concentrations. Fig. 1A shows the reaction time course of a typical kinetic experiment. The corresponding first-order kinetic plot is shown in Fig. 3. Rate constants for the two components were resolved graphically. Hb Binding and Release from Membranes. The binding and dissociation of Hb was studied in the same manner as that used for HbO2. Both reactions were biphasic, and the rate constants for the slow and fast phases were resolved graphically from the first-order kinetic plots (Figs. 4 and 5). As with HbO2, the binding rates were independent of ghost concentration and exhibited linear dependence on Hb concentration. The dissociation rates were independent on GAPD concentration. C2 4.0, I I I I I FIG. 3. Dissociation of the HbOmembrane complex, shown as first-order kinetic plot of the reaction. ph = 6.0; 5 mm phosphate; 200C; cell concentration (after mixing), 5 X 109 cells per liter; GAPD, 4.3 ysm; Hb, 0.04 ALM (tetramer). Other experimental conditions as in Fig. 1. The dashed line extends the linear portion of the slow phase. *, Experimental data; 0, fast phase resolved. W ZU2 20 W FIG. 4. Kinetics of Hb binding to erythrocyte membranes, shown as a semilogarithmic plot. ph 6.0; 5 mm phosphate; 200C; 0.3 mm dithionite; cell concentration, 5 X 109 cell per liter; Hb, 8.7,gM after mixing. The curve is resolved into two first-order components. The dashed line extends the linear portion of the slow phase. *, Experimental data; 0, fast phase resolved. DISCUSSION In the combination reactions studied here, the amount of Hb (1-100 mm) was much greater than that of Hb binding sites (0.1 mm). Therefore, the concentration of Hb was depleted only minimally during the reaction which can be considered as a pseudo-first-order process. The failure of the concentration of the cells to influence the rate of reaction is based upon the difference in locations of the reactants. In the reaction under study, Hb in the water phase is in equilibrium with the second reactant, the membrane sites (Ms), that are located in the solid membrane phase. The concentration of Ms, measured as sites per unit area, is a constant value which depends only on the membrane organization and is independent of the number of cells. The rate of reaction is thus independent of the "ghost" concentration. The elution of spectrin-actin complex, as occurs in the preparation of inside-out ghosts, is expected to change the organization of the membrane constituents and hence may alter the density of binding sites. We therefore elected to utilize preparations of open ghosts, which retain the major part of the spectrin-actin complex for the kinetic studies (9, 10). Because the same type of ghost preparation was used for our earlier steady-state studies of Hb binding, steady-state and kinetic data could be compared. The data show that the relative proportions of the two phases and the corresponding rate constants did not depend on Hb concentration. It therefore appeared that the binding rates and the biphasic pattern of the first-order plots did not result from differences in binding rates of dimeric and tetrameric Hb because, in the range of Hb concentration utilized in these studies,

7150 Biochemistry: Shaklai and Sharma 0 3.0 2.0 1.0 2.0 3.0 FIG. 5. Dissociation of the Hb membrane complex, in a first-order kinetic plot. ph = 6.0; 5 mm phosphate; 0.

4 7150 Biochemistry: Shaklai and Sharma FIG. 5. Dissociation of the Hb membrane complex, in a first-order kinetic plot. ph = 6.0; 5 mm phosphate; 0.3,M dithionite; 20 C; cell concentration, 5 X 109 cells per liter; GAPD, 5.2 im; Hb, 0.05 tm (tetramer). All concentrations are after mixing. The dashed line extends the linear portion of the slow phase. *, Experimental data; *, fast phase resolved. the proportion of dimer varies from 80% to a negligible percentage of the total Hb. The biphasic reaction time course observed for the reaction of Hb, which is almost completely tetrameric even in the micromolar concentration range (11), provides additional evidence against a difference in combination rates of Hb dimers and tetramers. Nor is the heterogeneity of the first-order kinetic plots related to an approach to equilibrium or to "local" depletion in Hb concentration because the ratio of the zero time amplitudes of the two phases was independent of Hb concentration. We suggest that the biphasic nature of the reaction time course represents heterogeneity in the nature of binding sites available for Hb on the membrane. The fast phase in the combination and the slow phase in the dissociation reactions of HbO2 and Hb contribute the same fraction (60%) of total fluorescence change in the reactions under widely varying conditions of Hb concentrations. These rates, then, are related to the same equilibrium reaction; the remaining two rate constants-i.e., slow "on" rate and fast "off" rate-represent the other equilibrium reaction. The kinetic Proc. Natl. Acad. Sci. USA 77 (1980) constants and the corresponding equilibrium constants calculated from them are given in Table 1. The kinetic pattern of Hb qualitatively resembles that of HbO2, but a significant quantitative difference is observed in the reaction rates. The "on" rates of fast and slow phases of Hb are much slower and the "off" rates are somewhat faster than the corresponding rates of HbO2. The equilibrium constants, calculated from the kinetic rate constants for the two sites, differ by an order of magnitude for HbO2 as well as for Hb. The equilibrium constants calculated from the kinetic and from previous steady-state experiments agree within experimental error. From the steady-state fluorescence studies, only the binding constant for Hb binding to the high-affinity sites could be calculated (1). The low-affinity sites were therefore characterized by a centrifugation method (2). If all bound Hb molecules have the same quenching potential, then from the number of lowaffinity sites (3-6 times larger than those of the high-affinity bound Hb) and the binding constant, some quenching of the fluorescence intensity related to the low-affinity sites should be observed. No such quenching was noted in the steady-state studies. Even in kinetic studies, in which much higher concentrations of Hb could be used, only 40% of the fluorescence intensity was quenched by Hb binding at low-affinity sites at full saturation with Hb; the calculations require 75-86% quenching, assuming the same energy transfer characteristics for the two types of sites. Because the spectral properties are the same for all Hb molecules and the same random orientation of Hb to fluorescence probe molecules is assumed for any bound Hb, the discrepancy noted above is probably related to the difference in the average distances from Hb to the probe at the two kinds of sites (1). The high-affinity sites, which exhibit more quenching than the low-affinity sites, are thought to be the cytoplasmic fraction of band 3 proteins (12, 13). The distance separating Hb from probe could result from deeper penetration of membrane by Hb molecules or, alternatively, Hb bound deep in the channel of band 3. At present we cannot distinguish between those two possibilities. The affinity of Hb for the membrane was low for both types of sites, and its binding could be measured in steady-state experiments only by comparing fluorescence quenching of HbO2 and Hb. This required calculations of energy transfer parameters for each and extrapolations to obtain values for fluorescence quenching at infinite time (3). Such calculations are subject to errors. The data obtained in the kinetic experiments were direct observations and free from the uncertainties of steady-state experiments. The low-affinity constant we have calculated here for Hb is of the order of 104 M-1 and could not have been measured by steady-state fluorescence quenching method. Because the relative proportions of high- and low-affinity components in the reactions of HbO2 and Hb were identical (60:40), it appears that the high- and low-affinity sites are the same for Hb and HbO2. Table 1. Kinetic and equilibrium constants Rate constants and zero-time fluorescence amplitudes, % of total k "on" % of k "off', % of Equilibrium constants (M-1 sec-1) X 106 reaction sec-1 reaction k "on"/k "off"' Steady state Oxyhemoglobin 9.0 ±3.0 60±2 0.07± :2 (1.5±0.8)X X108(1) 1.8 ± Q ± 2 (4.5± 2 )X X 106(2) Deoxyhernoglobin ±2 0.2±0.1 62±2 (5 ±3 )X106 2 X106(3) 0.09± ±2 2.0±0.4 38±2 (8 ±5 )X104

Biochemistry: Shaklai and Sharma It is interesting to note that the differences in the affinity of HbO2 and Hb for membrane arises mainly from the "on" rates.

5 Biochemistry: Shaklai and Sharma It is interesting to note that the differences in the affinity of HbO2 and Hb for membrane arises mainly from the "on" rates. Because the differences in net charge and size of HbO2 and Hb are minimal, the 10-fold difference in the "on" rates could not be due to differences in charge or diffusion rates. Although some of the large differences in quaternary structures of HbO2 and Hb are probably responsible, the specific reasons for the higher affinity of HbO2 are not clear to us at this time. This work was supported in part by Binational Science Foundation Grant No. 1879/79 and by Grants AM18781 and AM17348 from the National Institutes of Health. 1. Shaklai, N., Yguerabide, J. & Ranney, H. M. (1977) Biochemistry 16, Shaklai, N., Yguerabide, J. & Ranney, H. M. (1977) Biochemistry 16, Shaklai, N. & Abrahami, H. (1980) Biochem. Biophys. Res. Commun. 95, Proc. Nati. Acad. Sci. USA 77 (1980) Salhany, J. M. & Shaklai, N. (1979) Biochemistry 18, Fox, J. B. & Dadliker, W. B. (1956) J. Biol. Chem. 221, Coin, J. T. & Olson, J. S. (1979) J. Biol. Chem. 254, Kant, J. A. & Steck, T. L. (1973) J. Biol. Chem. 248, McDaniel, C. F., Fihy, M. E. & Tanner, M. J. A. (1974) J. Biol. Chem. 249, Steck, T. L. (1974) Methods Membr. Biol. 2, Marchesi, S. L., Steers, E., Marchesi, V. T. & Tillack, T. W. (1970) Biochemistry 9, Ib, S. H. C., Johnson, M. L. & Ackers, G. K. (1976) Biochemistry 15, Salhany, J. M., Cordes, R. A. & Gaines, E. D. (1980) Biochemistry 19, Sayari, M. & Schuster, T. M. (1980) Fed. Proc. Fed. Am. Soc. Exp. Biol. 39, 1916 (abstr.).

BINDING AND THE CONFORMATIONAL CHANGE OF HEMOGLOBIN

THE RELATION BETWEEN CARBON MONOXIDE BINDING AND THE CONFORMATIONAL CHANGE OF HEMOGLOBIN CHARLES A. SAWICKI AND QUENTIN H. GIBSON, Section of Biochemistry, Molecular and Cell Biology, Cornell University,