©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of the Dodecamer Subunit in the Dissociation and Reassembly of the Hexagonal Bilayer Structure of Lumbricus terrestris Hemoglobin (*)

(Received for publication, November 21, 1995; and in revised form, January 26, 1996)

Pawan K. Sharma (1)(§) Askar R. Kuchumov (1) Geneviève Chottard (2) Philip D. Martin (1) Joseph S. Wall (3) Serge N. Vinogradov (1)(¶)

From the  (1)Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, the (2)Laboratoire de Chimie des Métaux de Transition, URA 419, Université Pierre et Marie Curie, 75252 Paris, France, and the (3)Biology Department, Brookhaven National Laboratory, Upton, New York 11973

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The dissociation of the 3500-kDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris upon exposure to Gdm salts, urea and the heteropolytungstates [SiWO] (SiW), [NaSb(9)WO] (SbW) and [BaAs(4)WO] (AsW) at neutral pH was followed by gel filtration, SDS-polyacrylamide gel electrophoresis, and scanning transmission electron microscopy. Elution curves were fitted to sums of exponentially modified gaussians to represent the peaks due to undissociated oxyHb, D (200 kDa), T+L (50 kDa), and M (25 kDa) (T = disulfide-bonded trimer of chains a-c, M = chain d, and L = linker chains). OxyHb dissociation decreased in the order GdmbulletSCN > GdmbulletCl > urea > GdmbulletOAc and AsW > SbW > SiW. Scanning transmission electron microscopy mass mapping of D showed 10-nm particles with masses of 200 kDa, suggesting them to be dodecamers (a+b+c)(3)d(3). OxyHb dissociations in urea and GdmbulletCl and at alkaline pH could be fitted only as sums of 3 exponentials. The time course of D was bell-shaped, indicating it was an intermediate. Dissociations in SiW and upon conversion to metHb showed only two phases. The kinetic heterogeneity may be due to oxyHb structural heterogeneity. Formation of D was spontaneous during HBL reassembly, which was minimal (leq 10%) without Group IIA cations. During reassembly, maximal (60%) at 10 mM cation, D occurs at constant levels (15%), implying the dodecamer to be an intermediate.


INTRODUCTION

The giant, hexagonal bilayer (HBL) (^1)extracellular Hbs and chlorocruorin of annelids and vestimentiferans are 60 S proteins with an acidic isoelectric point, high cooperativity of oxygen binding, and a characteristically low iron and heme content, about two thirds of normal(1, 2, 3, 4) . They represent in many ways a summit of complexity for structures containing globins(5) . The most extensively studied Hb is that of the common North American earthworm Lumbricus terrestris. Although it has been the subject of numerous studies since Svedberg determined its mass by centrifugation in 1933, the molecular architecture of this complex of 180 polypeptide chains remains uncertain in the absence of a crystal structure. An early SDS-PAGE study showed that it consisted of at least six subunits(6) , four of which were globins, comprising a monomer subunit M (7) and a disulfide-bonded trimer T(8) , the remainder being linkers, chains of 24-32 kDa. The amino acid sequences of the T and M subunits have been determined(9, 10) . Although only three linker chains were thought to exist(11) , only one of which had been sequenced (12) , a recent ESI-mass spectroscopy study provided a detailed inventory of all the constituent polypeptide chains and indicated the existence of four linker chains(13) . Here we report the results of a study of the dissociation and reassembly of Lumbricus Hb, which support the role of the dodecamer of globin chains [3T+3M] as a principal intermediate in both processes.


EXPERIMENTAL PROCEDURES

Materials

L. terrestris Hb was prepared as described previously in 0.1 M TrisbulletCl buffer, pH 7.0, 1 mM EDTA, 2 mM phenylmethanesulfonyl fluoride, from live worms collected around London, Ontario (Carolina Wholesale Bait Co., Canton, NC)(4, 6) . The concentration of the Hb was determined from the absorbance of the native form at 280 nm or of the cyanmet form at 540 nm(4) , employing the respective extinction coefficients, 2.063 ± 0.032 mlbulletmgbulletcm and 0.442 ± 0.013 mlbulletmgbulletcm(13) . The Gdm salts were purum grade from Fluka AG (9470 Buchs, Switzerland) and urea was from Sigma. The heteropolytungstate salts KSiW (K(8)[SiWO].14H(2)O, 3239.2 Da), NaSbW (Na(18)[NaSb(9)WO].24H(2)O, 7178.4 Da) and AsW (Na[BaAs(4)WO].60H(2)O, 11,731.7 Da) were prepared according to Klemperer(14) .

Analytical Gel Filtration

Low pressure, isocratic gel filtration was carried out at room temperature (20 ± 2 °C) employing an FPLC system (Pharmacia Biotech Inc.) and 1 times 30-cm columns of Superose S12 or S6 (Pharmacia). Flow rate was 0.4 ml/min and the eluate was monitored at 280 nm. A constant amount of protein in a constant sample volume, 800 µg/200 µl, was loaded each time.

Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis in the presence of 0.1% SDS was carried out using the buffer system of Laemmli (15) and slab gels (1.5 mm times 10 cm times 8 cm) of 8-20% acrylamide. The gels were electrophoresed for 2-4 h, stained in 0.125% Coomassie Brilliant Blue R-250 in 45% methanol, 7.5% acetic acid, and destained in 25% methanol, 7.5% acetic acid.

Optical Spectrophotometry

The absorption spectra over the the 200-650 nm range were obtained using an OLIS (Bogart, GA) spectrophotometer employing a Hewlett Packard diode array detector or a Hitachi model 2000 spectrophotometer.

Fitting of Elution Profiles

The elution curves were either digitized on a Summagraphics Summasketch MM18 tablet using a Sigma Scan version 3.0 (Jandel Scientific, Corte Madera, CA) or acquired using the Easyest System 8 (Keithley Instruments, Inc., Rochester, NY) and an IBM PC/386 computer. The elution curve was then fitted as a sum of four EMGs, each representing the undissociated Hb (HBL) and peaks D, T+L, and M, employing least squares minimization (Peak Fit version 2.0, Jandel Scientific). The EMG function is a convolution of a gaussian and a decreasing exponential and is known to represent well the shape of chromatographic elution peaks(16, 17, 18) ,

where a(0) is the amplitude, a(1) is the center, a(2) is the width of the gaussian, and a(3) is the width of the exponential. The EMG is asymmetric with an exponential tail on the right side; the falloff rate of the tail is controlled by the parameter a(3). The areas of the individual peaks were plotted as percent of total versus time and fitted to sums of exponentials,

using PSI-Plot software (Poly Software International, Salt Lake City, UT) employing the Marquardt-Levenburg method. The acceptability of fits was judged by the absence of systematic trends in the plot of residuals with time.

Dissociation of Lumbricus OxyHb

The dissociating agent was dissolved in 0.1 M TrisbulletHCl buffer, pH 7.0, 1 mM EDTA, and Hb stock solution added to obtain the desired concentration, 3.6 mg/ml. The following dissociating agents were employed: urea, GdmbulletSCN, GdmbulletCl, GdmbulletOAc, SiW, SbW, and AsW. The dissociations of oxyHb at neutral pH and at pH 8.0 and 8.2 in 0.1 M TrisbulletCl buffer, 1 mM EDTA, were followed by FPLC at neutral pH.

Oxidation of Lumbricus Hb

The oxidation of Lumbricus oxyHb was effected by the addition of potassium ferricyanide (Fisher) or of sodium nitrite (Aldrich) at molar ratios relative to heme, ranging from 1 to 1000, in 0.1 M TrisbulletCl buffer, pH 7.0, 1 mM EDTA. The conversion of oxyHb to metHb was monitored using optical spectrophotometry over the 450-650 nm range and was complete in 5-20 min. The metHb solution was then immediately passed over a 1.5 times 20-cm Sephadex G-25 column to remove the oxidizing agent, and the progress of the dissociation measured using FPLC at neutral pH.

Reassembly of HBL Structure from Completely Dissociated OxyHb

Lumbricus Hb (30-60 mg/ml) was dissociated in the presence of 4-8 M urea in 0.1 M TrisbulletCl buffer, pH 7.0, 1 mM EDTA at room temperature. The completeness of the dissociation to T+L+M was checked by FPLC, urea was removed by dialysis against 2 times 1.5 liters of TrisbulletCl buffer for 2 h and the reassociation followed by FPLC in the presence of Group IIA cations Mg, Ca, and Sr over the 0-50 mM range; the solutions were kept at 7 °C. Alternatively, the T+L+M fractions were obtained by preparative gel filtration of oxyHb exposed to 4 M urea, pooled, concentrated by pressure filtration using Centricon 10 concentrators (Amicon Division, W. R. Grace & Co., Danvers, MA), and its reassociation followed in the absence and presence of Ca.

STEM Imaging and Mass Measurement of Unstained Protein

The mass measurements were performed using the STEM at the Brookhaven National Laboratory(19) . Preparation of the unstained specimens with TMV fibers as internal mass standards was carried out as described by Kapp et al.(20) . Negative staining was with 0.5% (w/w) uranyl acetate. The STEM was operated at 40 kev, a dose level <10 e/0.1 nm^2 and a resolution of 0.25 nm. An interactive program (19) was used to select the electron micrographs on the basis of clean background and apparent quality of TMV fibers and protein particles; it computes the background and permits the operator to select the TMV segments for internal mass calibration and the particles for mass measurement. At least 3-5 good TMV segments are generally chosen to calculate the internal mass calibration. The individual particles are selected based on clean background around the particles and absence of visible flaws.


RESULTS

Dissociation of OxyHb by Urea and Guanidinium Salts

Elution Profile of the Dissociation Products

Fig. 1A shows a typical FPLC elution profile of oxyHb dissociated at neutral pH in the presence of 1.5m GdmbulletCl. In addition to the undissociated Hb (HBL), three peaks are observed, D, T+L, and M, at elution volumes corresponding to approximately 200, 60, and 25 kDa. The unreduced SDS-PAGE (inset) shows that the subunit content of undissociated Hb is similar to the native Hb, peak D consists of subunits T (54.5 kDa; chains a+b+c) and M (16.6 kDa, chain d), peak T+L is the envelope of unresolved peaks due to subunit T and the four linker subunits L (L1-L4: 24.1, 24.9, 27.6, and 32.1 kDa), and peak M is the monomer (the masses provided here are from a recent mass spectrometric study of the Hb; (13) ).


Figure 1: FPLC elution profile at 280 nm of partially dissociated Lumbricus oxyHb in 0.1 M TrisbulletCl buffer, 1 mM EDTA, pH 7.0. A, exposure to 1.5 m GdmbulletCl; B, exposure to 12.4 mM SiW. The insets show the unreduced SDS-PAGE of native Hb (lane 1) and the indicated fractions. The undissociated peak is labeled HBL, and the three dissociated peaks are the dodecamer D, the trimer and linker subunits T+L, and the monomer subunit M. The profiles were obtained with different columns. Note that in B peak M is overlapped by the SiW peak.



Fitting of Elution Profiles

Fig. 2A shows a representative fit of an FPLC elution curve with four EMG functions. The elution volumes of the four peaks remained unchanged throughout the course of the dissociation to ±3%, as did the fitted variables a(1) (peak position). Fig. 2B shows a similar fit of an elution curve obtained following reassociation of T+L+M in 10 mM Ca.


Figure 2: EMG fits to the elution profiles at 280 nm obtained by FPLC. A, dissociation of oxyHb in 1.22 m GdmbulletCl after 28 h; B, reassociation of completely dissociated oxyHb in 10 mM Ca after 48 h. In A, the buffer also contained 1 mM EDTA. The differences in the elution volumes for the same subunits is due to the use of different columns.



Dissociation of OxyHb at Zero Time

In these experiments, the oxyHb was loaded on the column right after mixing with the dissociating agent and subjected to FPLC. The time elapsed between mixing and complete penetration of the sample into the column was 110-120 s. Plots of percent dissociation versus the concentration of the dissociating agent are shown in Fig. 3A.


Figure 3: Zero time dissociation of Lumbricus oxyHb. A, percent undissociated oxyHb versus concentration of GdmbulletSCN, GdmbulletCl, urea, and GdmbulletOAc. B, percent undissociated oxyHb (HBL) and peaks D, T+L, and M versus concentration of GdmbulletSCN and GdmbulletOAc. Each species was determined by resolution of FPLC profiles using the EMG function and is expressed as percent of total area.



Fig. 3B shows the relative percent of the four peaks as a function of increasing concentrations of GdmbulletSCN and GdmbulletOAc. The relative proportion of the dodecamer D is much less in GdmbulletSCN than in GdmbulletOAc, the weakest dissociating agent.

Time Course of OxyHb Dissociation in 4 M Urea and the Effect of Ca

Fig. 4A shows the time course of oxyHb dissociation in 4 M urea; although it is almost complete within 2 h, peak D remains constant indicating its stability in 4 M urea. Fig. 4B shows the time course of oxyHb dissociation in 4 M urea in 2.5 mM Ca; it can be fitted as the sum of two exponentials. However, since substantial dissociation occurs within the dead time of the FPLC method (2 min), there appear to be at least three separate dissociation processes with apparent tapprox leq1 min, 1 h, and 50 h. Fig. 4C illustrates the effect of [Ca] on the dissociation of oxyHb in 4 M urea after 144 h.


Figure 4: Time course of Lumbricus oxyHb dissociation in 0.1 M TrisbulletCl buffer, pH 7.0. A, in 4 M urea and 1 mM EDTA; B, in 4 M urea and 2.5 mM Ca. The dotted lines show the two exponential functions fitted with the resulting residual below. C, in 4 M urea after 144 h as a function of [Ca].



Kinetics of OxyHb Dissociation

Fig. 5A shows the time course of oxyHb dissociation in 1.75 m urea and Fig. 5(B-D), show the corresponding time courses of peaks D, T+L, and M, respectively. The insets show the initial phases. The points shown represent averages of values determined in three separate experiments. For all the processes shown in Fig. 5and for oxyHb dissociation in 1.22 m GdmbulletCl (results not shown), at least three exponentials were necessary to obtain a good fit as judged by the absence of any trends in the plots of residuals versus time provided at the bottom of each panel. Table 1summarizes the amplitudes and kinetic constants determined from the fits.


Figure 5: Time course of Lumbricus oxyHb dissociation in 1.75 m urea in 0.1 M TrisbulletCl buffer, pH 7, 1 mM EDTA. A, peak HBL; B, dodecamer; C, T+L subunits; D, M subunit, expressed as percent of total area. The insets show the dissociation over the first 200 h. The fits shown are to the sum of the three exponentials together with the resulting residuals. Note that the dodecamer reaches a maximum after 250 h (B) and then decreases and the absence of any induction period in the formation of peaks T+L and M (C and D, respectively).





STEM Imaging and Mass Mapping of Undissociated OxyHb and Peak D

Fig. 6shows views of unstained, cryolyophilized undissociated oxyHb obtained at 11% (A and C) and 89% (B and D) dissociation, respectively. Fig. 7A shows a histogram of the STEM masses of the complete HBL structures observed at 89% dissociation. Although the mean mass, 3540 ± 260 kDa (n = 120), is similar to the value 3560 ± 130 kDa obtained previously for native Hb(13) , the distribution of masses is more asymmetric at the lower end.


Figure 6: STEM images of Lumbricus Hb undissociated peak obtained by FPLC after exposure to 0.25 M GdmbulletSCN (A and C) and 0.6 M GdmbulletSCN (B and D) and of peak D obtained by FPLC at neutral pH, subsequent to exposure to 4.12 mM SiW (E) and pH 8.3 (F). All samples were in 0.05 M PIPES, pH 7.0. C and D represent 2.5-fold magnifications of selected areas from A and B, respectively. The extent of dissociation of the Hb was 10% in A and C and 89% in B and D. The scale bar in F represents 50 nm in C-F and 125 nm in A and B. Note the presence of deficient HBL structures lacking and in A-D.




Figure 7: Histograms of STEM masses of unstained, cryolyophilized specimens of the undissociated (HBL) peak obtained by FPLC of Lumbricus oxyHb dissociated in the presence of GdmbulletSCN to the extent of 89% of total (A) and of peak D fractions obtained by FPLC subsequent to exposure to SiW at neutral pH (B) and at pH 8.3 (C). They correspond to the STEM images shown in Fig. 6, panels B, E, and F, respectively.



Fig. 6(E and F) shows typical views of unstained, cryolyophilized peak D obtained by dissociation in SiW and at pH 8.3, respectively; the observed particles are 10 nm in diameter and histograms of the STEM masses within the range 150-250 kDa (Fig. 7, B and C) had corresponding mean masses of 200 ± 26 kDa and 195 ± 21 kDa, respectively.

Dissociation of OxyHb by Heteropolytungstates, at Alkaline pH and upon Conversion to MetHb

The complex heteropolytungstate anions SiW, SbW, and AsW are known to form 1:1 complexes with metMb at neutral pH with association constants in the 10^5 to 10^6M range and concomitant formation of hemichrome type visible absorption spectra (21) . All three dissociate oxyHb; a typical elution curve is shown in Fig. 1B. Fig. 8(A and B) shows the time courses of dissociation in 4.12 mM and 12.4 mM SiW, together with the fits to sums of two exponentials.


Figure 8: Time courses of dissociations in 0.1 M TrisbulletCl buffer, 1 mM EDTA. A and B, OxyHb at pH 7.0 in 4.2 mM and 12.6 mM SiW. C and D, OxyHb at pH 8.0 and 8.2, respectively. D and E, MetHb, following oxidation of oxyHb with K(6)Fe(CN)(6) and NaNO(2), respectively, and their removal by gel filtration. The exponential fits are shown as dotted lines together with the plots of residuals versus time below each panel.



It is well known that HBL Hbs dissociate at geqpH 8(7, 22) . Fig. 8(C and D) shows the time courses of dissociation at pH 8.0 and 8.2. Again, it is evident that a third, rapid phase occurs within the dead time of the FPLC (2 min). Thus, there appear to be three dissociation processes with tapprox leq1 min, 2-22 h, and 50-1200 h.

An early observation by Ascoli et al.(23) suggested that oxidation of earthworm Hb led to the dissociation of its quaternary structure. We reinvestigated this phenomenon because Lumbricus oxyHb was slowly altered to the met form during the dissociations in urea and GdmbulletCl. Fig. 8(E and F) shows the time courses of dissociation following the conversion of oxyHb to metHb and the removal of oxidant by gel filtration.

The fitted parameters for all the dissociations are provided in Table 1.

Reassembly of HBL Structure

Fig. 9shows some representative results obtained with the reassembly of HBL structures from completely dissociated oxyHb. In the absence of Group IIA cations, reassembly of was limited, generally much less than 10%. However, in the first 24 h, there is a spontaneous formation of dodecamer as illustrated in Fig. 9A. The same result is also observed in Fig. 9B, which depicts the time course of reassembly to HBL in 5 mM Mg. Fig. 9C illustrates the effect of cation concentration on the extent of HBL reassembly, and Fig. 9D shows that although there may be differences in the extent of reassembly achieved initially, the final [HBL] is remarkably similar for all three cations after 200 h.


Figure 9: Reassembly of HBL structure following complete dissociation of Lumbricus oxyHb (absence of HBL) in 8 M urea in 0.1 M TrisbulletCl buffer, pH 7, in the absence and presence of group IIA cations. A, time course of reassembly in 1 mM EDTA; note that mainly subunit D is formed with 1% HBL. B, time course of reassembly in 5 mM Mg. C, reassembly at 240 h versus [Ca]. D, time courses of reassembly in 10 mM Ca, Mg, and Sr.



Reassociation, starting with peaks T+L and M isolated by gel filtration of oxyHb dissociated in 4 M urea, shows that a spontaneous reassociation of T and M to about 20% D had occurred within 6 h prior to the first FPLC (Fig. 10), even though reassembly to the HBL was almost nonexistent (1%). Fig. 10also shows the reassembly time courses in 2.5 mM and 10 mM Ca; although the relative contents of T and M declined steadily, the level of peak D remained fairly constant at 10-15%. STEM images of unstained HBL[T+L+M] are indistinguishable from those of native Hb, and the mass distributions are similar to those determined for native Hb(13) . The time courses of HBL reassembly could be fitted reasonably well with a single asymptotic exponential.


Figure 10: Time course of reassembly of HBL structure in 0.1 M TrisbulletCl buffer, pH 7, in 2.5 mM Ca (circles) and 10 mM Ca (squares). The curves represent least squares fits to asymptotic exponentials Y = 24 - 23exp(-0.23t) and 46 - 38exp(-0.46t), respectively. The empty squares represent the time course of D as percent of total during reassembly in 10 mM Ca; the curve represents a least squares fit to a single asymptotic exponential Y = 11 + 7.3exp(-0.2t).




DISCUSSION

A Dodecamer [3T+3M] Is Observed in All Dissociations of Lumbricus OxyHb

The dissociation of the HBL structure at neutral pH by Gdm salts and heteropolytungstate anions and at mildly alkaline pH ( Fig. 1and Fig. 3) provide remarkably similar pictures; a 200-kDa dodecamer D ([3T+3M]), deficient in linker subunits, is always formed in addition to the M, T, and L subunits. In particular, dissociation in the weakest dissociating agent, namely GdmbulletOAc (Fig. 3), shows that D accounts for about half of the initial dissociation products. The time course of dissociation in 4 M urea (Fig. 4A) also shows that D accounts for 40-50% of the dissociation products. In addition, it appears that D is fairly stable in the presence of 4 M urea, in agreement with earlier findings(24) .

Fig. 3summarizes the effect of urea and several Gdm salts on the dissociation of Lumbricus oxyHb determined by FPLC at zero time. The order of decreasing effectiveness is GdmbulletSCN > GdmbulletCl > urea > GdmbulletOAc, with the order of the anions in line with the well known Hoffmeister series(25, 26) .

The order of increasing effectiveness of the three heteropolytungstates, SiW < SbW < AsW, appears to be correlated with their total charge and mass, -8 (3239 Da), -18 (7178 Da), and -27 (11,732 Da), respectively, and not with the surface charge density. Although SiW is spherical, SbW is a trigonal pyramid, and AsW is a parallelliped, the charge per unit area is approximately the same: -1.8, -2.0, and -2.1/100 Å^2, respectively(27) .

Effect of Ca on Urea Dissociation of OxyHb

Ca exerts a markedly protective effect on the quaternary structure of oxyHb in the presence of 4 M urea (Fig. 4). Although dissociation is almost complete (95%) after 2 h in 4 M urea (Fig. 4A), even 2.5 mM Ca reduces dissociation to 75% after 144 h in 4 M urea (Fig. 4B). The maximum protective effect is reached at [Ca] approx 10 mM (Fig. 4C). Alkaline earth (Group IIA) cations are known to stabilize the HBL structure of annelid Hbs with respect to dissociation at alkaline pH(22, 28, 29) , at acid pH(30, 31) , as well as thermal unfolding and autoxidation(32) . In some cases, such as Amphitrite Hb (33) and Myxicola chlorocruorin (34) , Ca is necessary for maintaining the HBL structure even at neutral pH.

The Kinetic Heterogeneity of Lumbricus OxyHb Dissociation

Our results show that dissociation of oxyHb followed by FPLC over several weeks is not accompanied by alteration in the properties of either the starting material or the products. 1) The elution volumes of the undissociated Hb (HBL) and of the products of its dissociation (peaks D, T+L, and M) remain unaltered. 2) The subunit compositions of all the peaks as judged by SDS-PAGE remain unchanged. 3) The STEM images of the HBL peak at an early (10%) and a late stage of dissociation (89%) indicate no major alterations in dimensions (Fig. 6, A-D). Furthermore, the STEM mass distribution at 89% dissociation (Fig. 7A) compared to that of the native Hb (13) exhibits only a slight asymmetry at the low end, probably due to the presence of a relatively small number of ``deficient'' HBLs, missing and of the HBL structure that can be observed in Fig. 6(A-D).

The time courses of oxyHb dissociation in 1.75 m urea (Fig. 5A) and 1.22 m GdmbulletCl at neutral pH can be satisfactorily represented as the sum of three first-order processes with tapprox 1-2 h, 30-50 h, and 400-500 h (Table 1). Fig. 4also shows that there are at least three processes occurring in the dissociation of oxyHb in 4 M urea in the absence and presence of Ca.

Three first-order processes are also observed in oxyHb dissociation at alkaline pH, tapprox leq1 min, 2-20 h, and 50-1200 h (Table 1). OxyHb dissociation in the presence of SiW (Fig. 8, A and B) can be fitted with two first-order processes, tapprox 10-40 h and 400-1300 h (Table 1). The latter values correspond roughly to the tfor the two slower dissociation processes in urea and GdmbulletCl and at alkaline pH.

Two points must be considered before discussing possible mechanisms for the dissociation of Lumbricus oxyHb. 1) Whether slow oxidation of oxyHb to metHb could be responsible for one of the dissociation processes observed. MetHb dissociation (Fig. 8, E and F, and Table 1) consists of two phases: a small (10%) initial dissociation (t 2 h), followed by a dissociation that is slower by more than 1 order of magnitude than the slowest phase of the oxyHb dissociations (tapprox 13,000-35,000 h versus 50-1300 h). Hence, dissociation due to metHb formation can be neglected. 2) Can the dissociation of the oxyHb be accompanied by a partial disruption of the tertiary and secondary structures of the globin subunits? It is known that myoglobin does not evince any conformational alterations at urea concentrations less than 5 M(35, 36) . Hence, it is unlikely that 4 M urea affects either the M or the disulfide-bonded T subunit.

Possible Mechanisms of OxyHb Dissociation

Several simultaneous dissociations of a HBL structure can be envisaged (Fig. 11A): to D+L(1) , T+L+M(2) , D+T+L+M(3) , and (4) dissociation of D from (1) and (3) to T+M. STEM images of Hb at 11% and 89% dissociation (Fig. 6, A-D) show the presence of deficient HBLs, partially dissociated Hb particles lacking and of the HBL structure (represented schematically in Fig. 11B). We do not know whether these deficient HBLs are intermediates or not. The time course of the appearance of D, which reaches a maximum in the the initial 10% of the dissociation and decreases thereafter (Fig. 5B), is consonant with the formation of deficient HBLs with concomitant formation only of D (Fig. 11B), being the initial stage of HBL dissociation. The latter is reminiscent of the time course of formation of an intermediate B in a simple set of consecutive first-order reactions A B C(37) . However, this simple scheme does not fit our results, since: 1) oxyHb dissociation can not be represented by a single exponential and 2) the time courses of T+L and M appearances (Fig. 5, C and D) do not exhibit an induction period and consequently, an inflection point in their curves, as does the appearance of the final product C in the foregoing model. The latter result suggests that processes(1) -(3) occur simultaneously.


Figure 11: Schematic representation of possible processes in the dissociation of Lumbricus oxyHb HBL structure (A), the formation of deficient HBLs missing and (B), and a simple two-step reassembly to HBL structures (C).



The dissociation of the dodecamer in the presence of urea and GdmbulletCl requires two exponentials for a satisfactory fit with tapprox 100-200 h and 2700-5000 h. (^2)The slower process has a tclose to that determined for the dissociation of the metdodecamer, which is about an order of magnitude faster than the dissociation of the metHb. It is likely that oxydodecamer dissociation (tapprox 100-200 h) occurs mostly in the later stages of oxyHb dissociation, following the accretion of peak D observed in the first 50-300 h (Fig. 5B).

There seem to be two simple explanations for the kinetic heterogeneity of oxyHb dissociation. 1) Since peak HBL, whose area is a measure of undissociated HBL structures, contains ``complete'' HBLs as well as the deficient HBLs lacking and of the structure, one explanation is that the observed three first-order processes reflect the dissociation of the complete and deficient HBLs. However, the STEM appearance and STEM mass distributions at a late stage of dissociation (Fig. 6, B and C, and 7A) indicate the presence of limited numbers of deficient HBLs. 2) Another possibility is that the native Hb consists of three unequal populations of HBL structures differing in their stabilities toward dissociation, each population of HBLs exhibiting its own rate of dissociation in the presence of a given concentration of the dissociating agent. In this view, the deficient HBLs are likely intermediates in the overall dissociations. Our results suggest that the initial, rapid oxyHb dissociation with tof leq1 min at alkaline pH and 1-2 h in 1.75 m urea and 1.22 m GdmbulletCl, which is not observed in the case of SiW, may be related to the ease of penetration into the Hb interior. The penetration of OH and its reaction, e.g. with salt bridges stabilizing some intersubunit contacts, should be much more rapid than the penetration by urea or GdmbulletCl and their binding to enough peptide groups and/or side-chain groups of the different subunits to effect a similar destabilization. This notion is consistent with the probable inability of the heteropolytungstates to penetrate into the Hb interior and the consequent occurrence of only two first-order processes (tapprox 10-40 h and 400-1300 h), comparable to the two slower processes observed in urea and GdmbulletCl and at pH 8.0 (tapprox 22-53 h and 400-1200 h, Table 1).

Role of the Dodecamer in HBL Structure Reassembly

Our results ( Fig. 9and Fig. 10) demonstrate that dodecamers are formed both in the presence and absence of the linker subunits L and in the absence and presence of Group IIA cations. Likewise, in the presence of Mg and Sr (Fig. 9B), but not Ca, there is an increase in D which occurs in the first 24 h, prior to the formation of any significant amount of HBL. These facts suggests that formation of the dodecamer precedes that of HBL and that the dodecamer is an obligatory intermediate in the reassembly of the HBL structure (Fig. 11C). In the presence of Ca (Fig. 10), the formation of HBL is accompanied by only a small decrease in D, the latter remaining at a fairly constant level between 10 and 15%. At optimum concentrations of Mg, Ca, and Sr, approx10 mM in all three cases (Fig. 9C), the extent of HBL reassembly reaches 50-60%, again indicating a dominant role for Group IIA cations in stabilizing the HBL structure.

In contrast to the kinetic heterogenity of HBL dissociation (Fig. 4, 5, and 8), the time course of HBL reassembly ( Fig. 9and Fig. 10) is readily fitted with a single asymptotic exponential. The first step of dodecamer formation (Fig. 11) appears to be relatively fast; hence, the observed process is likely to be the second step of dodecamer combination with linker subunits to form HBL structures (Fig. 11). Peaks intermediate between HBL and D occur in the elution profiles of reassociating mixtures (peaks I1 and I2 in Fig. 2B and Fig. 9B). At present, we do not know whether they are intermediates or reassembly-incompetent side-products.

Is Subunit Stoichiometry Constant in HBL Structures?

Native Lumbricus Hb examined by STEM mass mapping and sedimentation equilibrium, exhibits a fairly broad range of masses from 3200 to 3900 kDa(13) . HBL structures can be reassembled from T+L subunits(38) , and we have recently shown them to have STEM masses ranging from 2500 to 3600 kDa. (^3)Although the distribution is asymmetrical with a tail at lower masses than 3000 kDa, the surprising observation is that a considerable fraction of the masses are higher than 3000 kDa, the mass of native Hb, 3560 kDa, minus the contribution of the M subunit, 575 kDa(13) . A possible explanation is that there may occur extensive formation of ``pseudo-dodecamers'' consisting of 4T subunits instead of [3T+3M], which preserves the local 3-fold symmetry found in the dodecamer crystal(39) .

Furthermore, the recent three-dimensional reconstructions from cryoelectron microscopic images of Eudistylia chlorocruorin, Macrobdella Hb, Lumbricus Hb, and reassembled HBL missing one of the linker subunits of Lumbricus Hb by Lamy and collaborators (40, 41) (^4)demonstrate that all the HBL structures are very similar. An obvious explanation is that HBL structures may not require a fixed stoichiometry of globin and linker subunits. Hence, structural heterogeneity of Lumbricus Hb may lie at the heart of the kinetic heterogeneity of its dissociation.

Conclusion

The results presented here extend our earlier findings (24, 42) and provide conclusive evidence for the dodecamer [3T+3M] being the principal structural intermediate in the dissociation and reassembly of Lumbricus Hb.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK38674. The Brookhaven STEM Facility is supported by the United States Department of Energy and by the National Institutes of Health Biotechnology Resources Branch Grant RR01777. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Cardiology, School of Medicine, University of Utah, Salt Lake City, UT 84109.

To whom all correspondence should be addressed. Tel.: 313-577-1501; Fax: 313-577-2765; svinogr{at}cms.cc.wayne.edu.

(^1)
The abbreviations used are: HBL, hexagonal bilayer; Hb, hemoglobin; Mb, myoglobin; KSiW, potassium undecatungstosilicate, K(8)[SiWO].14H(2)O; NaSbW, sodium heni-cosatungstononaantimonate (III), Na(18)[NaSb(9)WO].24H(2)O; NaAsW, sodium tetracontatungstotetra-arsenate(III), Na[BaAs(4)WO].60H(2)O; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; EMG, exponentially modified gaussian; Gdm, guanidinium; SNC, thiocyanate; OAc, acetate; STEM, scanning transmission electron microscopy; PIPES, piperazine-N,N`-bis[2-ethanesulfonic acid]; TMV, tobacco mosaic virus.

(^2)
P. K. Sharma and S. N. Vinogradov, unpublished observations.

(^3)
A. R. Kuchumov, S. N. Vinogradov, and J. S. Wall, unpublished observations.

(^4)
F. De Haas and J. Lamy, unpublished observations.


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