Proc. Nati. Acad. Sci. USAVol. 73, No. 5, pp. 1581-1585, May 1976Biochemistry

Nuclear magnetic resonance study of heme-heme interaction inhemoglobin M Milwaukee: Implications concerning the mechanismof cooperative ligand binding in normal hemoglobin*

(1H nuclear magnetic resonance/allosteric models/oxygenation of Hb/valency hybrid Hb)

LESLIE W.-M. FUNGt, ALLEN P. MINTONt, AND CHIEN HOt §Department of Life Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260; and * Laboratory of Biophysical Chemistry, National Institute of

Arthrtis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014

Communicated by Julian M. Sturtevant, March 5, 1976

ABSTRACT Hemoglobin M Milwaukee (867E11 Val- Glu)is a naturally occurring valency hybrid containing two perma-nently oxidized hemes in the #-chains. In this mutant, the twoabnormal #-chains cannot combine with oxygen, whereas thetwo a-chains are normal and can combine with oxygen coop-eratively with a Hill coefficient of approximately 1.3. High-resolution proton nuclear magnetic resonance spectroscopy at250 MHz has been used to investigate the hyperfine shiftedresonances of the abnormal ferric #-chains ofHbM Milwaukeeover the spectral region from -30 to -60 parts per million fromwater at pD 7 and 300. These resonances are found to changeas a function of oxygenation of the normal a-chains in thismutant hemoglobin. The proton resonance spectra of the ferricft-hemes of partially oxygenated Hb M Milwaukee can be de-scribed as an appropriately weighted average of the spectra ofzero-, singly-, and doubly-oxygenated species. The nuclearmagnetic resonance spectrum of the singly-oxygenated specieshas been calculated by a method employing least-squaresanalysis of the spectra of partially oxygenated HbM Milwaukeeat several values of oxygen saturation. It is different from thatof fully deoxy or fully oxygenated HbM Milwaukee and cannotbe described as an average of the spectra of the fully deoxy andoxy species. These results are not consistent with a two-structuremodel for the oxygenation of this mutant protein. In view of thesimilarities between normal adult hemoglobin and HbM Mil-waukee, it is suggested that a two-state concerted allostericmodel does not provide an adequate description of the struc-ture-function relationships in normal hemoglobin.

Despite the fact that our knowledge of the structure-functionrelationship is much more advanced in hemoglobin (Hb) thanin other proteins, we still do not fully understand the detailedmolecular mechanism for the cooperative oxygenation of thisprotein molecule. The affinity for heme-ligand of an unligatedsubunit of hemoglobin is a function of the number of othersubunits to which heme-ligand is already bound. This phe-nomenon, commonly known as heme-heme interaction, hasbeen the subject of intensive research, and numerous attemptshave been made to describe the underlying mechanism ofheme-heme interaction in terms of the structure of hemoglobin(for example, see ref. 1). Most of these descriptions fall into oneof two classes of models, called two-state concerted (for ex-ample, see ref. 2) and sequential (for example, see ref. 3). Ac-

Abbreviations: Hb A, normal human adult hemoglobin; HbO2, oxy-

hemoglobin; HbCO, carbonmonoxyhemoglobin; NMR, nuclearmagnetic resonance; ppm, parts per million; Bis-Tris, [bis(2-hy-droxyethyl)amino]tris(hydroxymethyl)methane; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; T, low-affinity ("tense") conformation; R,high-affinity ("relaxed") conformation.* This paper was presented in part at the 20th Annual Meeting of theBiophysical Society, February 24-27, 1976, Seattle, Wash.

§ Address all inquiries and reprint requests to: Dr. Chien Ho, Depart-ment of Life Sciences, 378 Crawford Hall, University of Pittsburgh,Pittsburgh, Pa. 15260.

1581

cording to the two-state concerted description, a particular li-gated species (for example, hemoglobin with two oxygenmolecules bound) exists as an equilibrium mixture of low-af-finity (T) and high-affinity (R) conformations. Under ordinaryconditions, the fully deoxygenated molecule is presumed to existprimarily in the T conformation and the fully oxygenatedmolecule is presumed to exist primarily in the R conformation.The ligation of a subunit shifts the equilibrium between con-formations in favor of the high-affinity form, and thereby in-directly increases the equilibrium average affinity of remainingunligated subunits in the molecule. According to a sequentialdescription, the ligation of a subunit alters intersubunit inter-actions in such a manner as to destabilize the conformation ofa neighboring unligated subunit relative to the conformationwhich this subunit adopts upon ligation. In this manner the li-gation of one subunit directly increases the oxygen affinity ofits neighbors.

It is difficult to experimentally ascertain whether themechanism of heme-heme interaction is more appropriatelydescribed by a two-state concerted or sequential model, inas-much as many of the observed ligand-binding properties andligand-dependent structural changes may be accounted forequally well by both kinds of model. In large measure this is dueto the fact that because of the cooperative nature of the li-gand-binding process, partially heme-ligated intermediatespecies never account for more than a small fraction of the totalhemoglobin content, and it is only in the description of theseintermediates that the two classes of model may be operation-ally distinguished.

Synthetic valency hybrids have been used as models for un-derstanding the nature and roles of intermediate species in thecooperative oxygenation process. In these hemoglobin mole-cules, the heme groups in either the a- or ,8-chains are selec-tively oxidized, whereas the other hemes remain in the ferrousstate capable of binding 02 or CO (such as a2+/32 or a2/32+).They are generally assumed to be similar in structure to par-tially oxygen-ligated molecules of normal hemoglobin, such asa2°g#2 or a2(232 (4-14). The structural and functional propertiesof the mutant hemoglobins M, which are naturally occurringvalency hybrids, have also been studied extensively (15-22).One of these, Hb M Milwaukee, has the glutamyl residue at/367E11 substituted for the normally occurring valine (23).According to x-ray studies, the Eli residue is positioned directlyabove the heme group adjacent to the distal histidine, and thecarboxylate oxygen of the glutamyl residue binds to the ferric/3-iron as the sixth ligand (24). This substitution allows the /3-hemes to become oxidized in erythrocytes and the patientsuffers from methemoglobinemia (25). The oxygen affinity ofHb M Milwaukee is less than that of normal adult hemoglobin(Hb A) [at 10°, the oxygen pressure in equilibrium with half-

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saturated Hb, pso, = 21 torr (2.8 kPa) in 0.1 M phosphate at pH7.2, compared to 2.9 torr (390 Pa) for Hb A] and the Hill coef-ficient is about 1.3 (26).

In this preliminary communication, we wish to summarizesome of our recent findings on Hb M Milwaukee, in particularthe implications of the proton nuclear magnetic resonance(NMR) spectral changes as a function of oxygenation on themolecular mechanism for the oxygenation of this mutant pro-tein and of normal human adult hemoglobin. A preliminaryreport of our early 1H NMR study of this mutant was given in1972 (29). A detailed description of our 'H NMR studies of HbM Milwaukee will be published elsewhere (L. W.-M. Fung, A.P. Minton, T. R. Lindstrom, A. V. Pisciotta, and C. Ho, manu-script in preparation). (For recent reviews of our current 1HNMR studies of hemoglobins, see refs. 27 and 28.)

MATERIALS AND METHODSWhole blood containing Hb M Milwaukee was drawn from aheterozygous patient and treated with acid-citrate-dextroseto prevent coagulation. Hb M Milwaukee was isolated from theother hemoglobin components of the hemolysate, mainly HbA, by column chromatography using Bio-Rex 70 resins (17). TheNMR samples were prepared from the isolated Hb M Mil-waukee fraction by first stripping the hemoglobin moleculesof all remaining phosphates by chromatography on a SephadexG-25 column in 0.01 M Tris-HCl plus 0.1 M NaCl at pH 7.5 (30).The samples were exchanged with D20 by repetitive dilutionand then concentrated to the final concentration of about 15g/100 ml by an ultrafiltration membrane (UM-20E, Amicon).Deoxygenated samples were prepared by standard methodsused in this laboratory and were transferred to regular 5 mmNMR sample tubes (31). An oxygen pressure of about 2 atmo-spheres (200 kPa) was required to obtain a fully oxygenatedsample. Partially oxygenated samples were prepared by mixingappropriate amounts (by volume) of deoxy and oxy samples.HbM Milwaukee concentrations were determined spectro-

photometrically on a Cary 14 spectrophotometer at 620 nmusing a millimolar extinction coefficient (e) of 4.4 for (3+-hemeand at 540 nm using e = 13.4 for a-heme (32). Hydrogen ionconcentrations were determined on a Radiometer model 26 pHmeter equipped with a Beckman model 39030 combinationelectrode. Deuterium ion concentrations were estimated byadding 0.4 pH units to the pH meter readings for the D20 so-lutions (33).The 1H NMR spectra were obtained on the MPC-HF 250

MHz superconducting spectrometer interfaced with a Sigma-5computer (34). The signal-to-noise ratio was enhanced bymultiscan accumulation and by the NMR correlation technique(35). Proton chemical shifts were referenced with respect to theresidual water proton signal in the sample, which is 4.75 partsper million (ppm) downfield from the proton resonance of astandard, 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), at 30Q,the ambient temperature of the probe. Chemical shifts down-field from water are assigned negative values and are accurateto I1.0 ppm.

RESULTSBoth the deoxygenated and oxygenated samples of Hb MMilwaukee give NMR signals in the spectral region between-30 and -60 ppm downfield from water. These greatly shiftedresonances arise from hyperfine interactions of the unpairedelectrons of the ferric iron atom with protons on the porphyrinring (36., 37) and are associated exclusively with the 0+-hemesof Hb M Milwaukee, where the heme iron atoms are perma-nently oxidized. As shown in Fig. 1 (solid lines), in 0.1

a

b

c

d

e

FIG. 1. The 250 MHz proton nuclear magnetic resonance spectraof 15 g/100 ml HbM Milwaukee (,B67E11 Val - Glu) in D20 and 0.1M Bis-Tris at pD 7.0 and 300. The units are given in ppm from HDO.Ho represents the applied magnetic field, which increases from leftto right as shown in the figure. The solid lines are experimental spectraand the broken lines are calculated spectra based on a three-speciesmodel. These lowfield paramagnetic or hyperfine shifted resonancesare due to the abnormal ferric f-chains.

M [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane(Bis-Tris) buffer in D20 at pD 7.0, when the a-chains aredeoxygenated, distinct resonances are observed at about -48,-53, and -55 to -57 ppm. When the a-chains are fully satu-rated with 02 or CO, three major resonances at about -45, -52,and -57 ppm are present. Hereafter, we shall refer to spectraresembling that shown in Fig. la as type a and spectra resem-bling that shown in Fig. If as type f. Upon stepwise additionof oxygen to the a-chains, the g+-heme spectrum changesgradually from type a to type f, as shown in Fig. lb-e, with the-48 ppm resonance being replaced by the -45 ppm resonanceand the ratio of the intensity of the resonance at -55 to -57ppm to that of the resonance at -53 ppm decreasing fromgreater than one to less than one. In this series of spectra, Fig.le is of special interest. When the a-chains are about 80% sat-urated with oxygen, the -48 and the -45 ppm resonancescoexist with approximately equal intensities. This spectralfeature indicates that the protons responsible for these tworesonances interconvert slowly on the NMR time scale, i.e.,greater than 0.4 msec (for example, see ref. 38).

DISCUSSIONHb M Milwaukee provides an attractive system for the studyof heme-heme interaction in hemoglobin because it possessesthe following advantageous features not found in Hb A: (i) thereexists only one partially oxygenated intermediate species; (ii)cooperativity of ligand binding, while present, is sufficientlylow so that the singly-oxygenated intermediate makes up asignificant fraction of the total hemoglobin content at inter-mediate values of the oxygen saturation; and (iii) the electronicstructure or magnetic environment of the ferric Tl-hemes maybe monitored (by means of the ferric hyperfine shifted reso-

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nances) as a function of the oxygen saturation of the normalferrous a-hemes. In this manner one can directly observe thealterations at the oxygen binding sites on one type of subunit(,6+-hemes) which are associated with the binding of oxygento neighboring subunits (a-chains). In addition, the deoxy hy-perfine shifted 1H NMR spectra characterizing the oxygenbinding sites in the a-chains of Hb M Milwaukee indicate thatthese hemes bind oxygen and interact with their environmentsin a manner which is highly similar to that of the a-hemes innormal hemoglobin. Furthermore, the NMR spectra charac-terizing the tertiary (as indicated by the deoxy hyperfine andring-current shifted proton resonances) and quaternary (asindicated by the exchangeable proton resonances) structuresof Hb M Milwaukee indicate that the tertiary structure aroundthe a-heme pocket and the quaternary structure of the moleculeas a whole are highly similar to the-corresponding structuresin normal hemoglobin (detailed results to be published). Thus,we may be confident that the mechanism of the heme-hemeinteraction in Hb M Milwaukee is not qualitatively differentfrom that operating in normal hemoglobin.The results obtained from the studies of the partially oxy-

genated samples as shown in Fig. 1 offer a new insight into thenature of the structural changes accompanying oxygenationin Hb M Milwaukee as well as in Hb A. The major changes inthe spectra of #+-chains that accompany oxygenation of thea-chains are (i) the replacement of the -48 ppm peak in thedeoxy form by a -45 ppm peak in the oxy Hb M Milwaukee,and (ii) inversion of the intensity ratio of the peaks at about -57and -52 ppm. We believe that these two spectral changes re-flect changes in two different regions of the ,8+-chain hemeenvironment. Examination of the spectra of the partially ligatedspecies in Fig. 1 (solid lines) reveals that these two spectralchanges and the associated structural changes are asynchronous.At intermediate values of oxygen saturation, the hyperfineshifted resonance spectra of the f+-chains show some featureswhich are not found in the type a or type f spectra. Thus, theferric hyperfine shifted resonances of Hb M Milwaukee indicatethat the environment of the ferric T3-hemes is influenced by theoxygen saturation of the a-hemes. Sufficient quantities of or-ganic phosphates can partially or completely inhibit thestructural change in the 6+-chains accompanying the oxy-genation of the a-hemes (detailed results to be published).The spectrum of the singly-oxygenated intermediate (Fig.

2) can be obtained by the least-squares procedure which willbe fully described in a subsequent publication. Briefly, the ferrichyperfine shifted proton NMR spectrum of HbM Milwaukeeat each level of oxygen saturation was digitized to yield 370points (xa) where x is the chemical shift and a is the spectralamplitude. To a good approximation, the ferric hyperfineshifted spectrum of partially oxygenated HbM Milwaukee maybe considered to be an appropriately weighted average of threespectral contributions corresponding to the zero-, singly-, anddoubly-oxygenated species:

a(x,y) = fo(y)ao(x) + fi(y)al(x) + fXy)a,(x)where f (y) is the fraction of Hb M Milwaukee to which i oxy-gen molecules are bound at an overall fractional saturation, y.Thefi are analytical functions of y and the Hill coefficient. Thespectral amplitude of the singly-oxygenated species, al(x), isobtained from the above equation by a least-squares fit of thisequation to the data points obtained from the four experimentalspectra (y = 0.2,0.4, 0.6, and 0.8) as shown in Fig. 1(solid lines).Also plotted in this figure (broken lines) are spectra of partiallyoxygenated solutions of Hb M Milwaukee that are calculated

a

b

c

0- liganded\ (deoxy)

H0 -

FIG. 2. The 250 MHz proton nuclear magnetic resonance spectraof deoxy and oxy Hb M Milwaukee (a and c) and spectrum of thesingly-oxygenated intermediate of Hb M Milwaukee (b) calculatedas described in the text. Units and coordinate are the same as in Fig.1.

from the above equation, with the appropriate set of fi and thethree spectra in Fig. 2. These calculated spectra of partiallyoxygenated Hb M Milwaukee agree with the observed spectrato well within the uncertainty introduced by the experimentalerrors and a lack of precision in the input values of y and theHill coefficient. It should be pointed out that test calculationsshow that the variation of the Hill coefficient between 1.0 and1.4 has only a minor quantitative effect and no qualitative effectupon the results discussed here.One major goal of the present investigation is to determine

whether the changes which are observed to take place in thevicinity of the ,+-hemes when oxygen molecules are bound tothe a-chains are inconsistent with the predictions of either thetwo-state concerted or sequential class of models. Accordingto a two-state concerted model, the zero-, singly-, and doubly-oxygenated species of Hb M Milwaukee should exist as equi-librium mixtures of molecules in the T and R conformations.It may readily be shown that if this is the case, then the structureof singly-oxygenated Hb M Milwaukee should be describableas an equilibrium average of the structures of zero- and dou-bly-oxygenated Hb M Milwaukee. (This conclusion is validindependent of the values of the allosteric constants for zero-and doubly-oxygenated Hb M Milwaukee.) Let us assume thata given structure is associated with a single ferric hyperfineshifted spectral pattern. It is not necessary to assume that theconverse is true. Then the concerted two-state model predictsthat (i) the spectrum of 6+-chains of singly-oxygenated HbMMilwaukee will be an average of the corresponding spectra ofdeoxy (zero-oxygenated) and oxy (doubly-oxygenated) Hb MMilwaukee, and that as a consequence (ii) the spectra of 6B+-chains of HbM Milwaukee at any intermediate level of oxygensaturation will be an appropriately weighted average of thespectra of deoxy and oxy HbM Milwaukee. Comparison of thespectra of partially oxygenated Hb M Milwaukee shown in Fig.1, the calculated spectrum of singly-oxygenated Hb M Mil-

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- 56.9

" T "(deoxy)

'O.9T +0.1 R

' 0.8T+0.2R

' 0.7T+0.3R

0.6T+0.4R

0.5T+0.5R

i 0.4T+0.6R

0.3T+0.7R

0.2T+0.8R

i0.iT+0.9R

(oxy )

Ho -

FIG. 3. Calculated proton nuclear magnetic resonance spectraof partially oxygenated Hb M Milwaukee according to a two-statemodel. Units and coordinate are the same as in Fig. 1.

waukee in Fig. 2, and the simulated two-state spectra shownin Fig. 3 clearly show that these predictions are not even ap-

proximately correct.The spectrum of singly-oxygenated Hb M Milwaukee shows

a broad peak extending from about -52 to -57 ppm, whichappears to be intermediate between the corresponding struc-tural features in zero- and doubly-oxygenated Hb M Milwaukee(Fig. 2). However, the spectrum of singly-oxygenated speciesalso shows a peak at -48 ppm that is identical, within experi-mental error, to that found in the spectrum of deoxy Hb MMilwaukee. We note that the ferric hyperfine shifted protonspectrum of singly-oxygenated Hb M Milwaukee containsspectral contributions from two heme groups which are nec-

essarily equivalent in fully deoxygenated or fully oxygenatedhemoglobin, but which are geometrically or conformationallynonequivalent relative to the singly-oxygenated heme of thismolecular species, and therefore may have nonidentical spectraas well. Thus, the ferric hyperfine shifted spectrum of singly-oxygen ligated Hb M Milwaukee shown in Fig. 2 admits of twopossible interpretations. First, if the two,+-hemes have iden-tical spectra, then one part of the environment of each O+-hemehas a structure intermediate between that of deoxy and oxy HbM Milwaukee, whereas another part of the ft+-heme environ-ment has a structure closely resembling that of deoxy Hb MMilwaukee. Second, if the two (.+-hemes have nonidenticalspectra, then it is possible that one of the (+-hemes in singly-oxyHb M Milwaukee is in an environment which completely re-

sembles that in deoxy Hb M Milwaukee, while the other f+-heme is in an environment which only partially resembles thatin deoxy Hb M Milwaukee. Neither is consistent with any

straightforward structural interpretation of the concertedtwo-state model. Both of these possibilities are, on the otherhand, consistent with a general sequential description whichallows for nonequivalence of a- and 3-subunits and the inter-actions between them. One example of such a description is theextended Coryell model (14).

There are several differences between the present NMRresults and those reported by Shulman and coworkers on syn-thetic valency hybrids (a2+CN2 and a232+CN) and HbM Iwate(a87F8 His -- Tyr) (7, 11, 12, 21, 43). Generally, paramagne-tically shifted NMR peaks of protons on and near the hemes inthe low-spin ferric and high-spin ferrous states as well as thering-current shifted resonance at about +1.8 ppm from DSS(or about +6.4 ppm from HDO at room temperature) wereused to monitor structural changes around the hemes. In ad-dition, an exchangeable proton resonance at about -14 ppmfrom DSS (or about -9.4 ppm from H20) has been used as aprobe for the deoxy quaternary structure. The NMR results ofShulman and coworkers have provided some insight into thestructure-function relationships in hemoglobin. However, forthe following reasons, they are of limited value as evidence tosupport an allosteric model. (i) Essential data on spectralchanges at intermediate values of 02 or CO saturation were notreported. (ii) The hyperfine shifted proton resonances associ-ated with deoxy and cyanomet hemes overlap with each otherin the spectral region examined (-10 to -25 ppm from DSS or-5 to -20 ppm from HDO). (iii) Synthetic valency hybrids aregenerally not very stable and thus it is difficult to obtain reliabledata for experimental measurements requiring a long periodof time.The findings discussed above indicate that during the course

of oxygenation the environment of at least one of the two ,+-hemes in Hb M Milwaukee takes on a structure which is inter-mediate between that of deoxy and oxy states, and show thatinformation exists at the #+-hemes in Hb M Milwaukee as towhether zero, one, or two oxygen molecules are bound to thea-hemes. It could be argued that there is no direct relationshipbetween the observed spectral (and associated structural)changes and the oxygen affinity of the #-chain binding site ina normal hemoglobin molecule. Therefore this discovery doesnot, in and of itself, conclusively establish the existence of morethan two affinity states in Hb M Milwaukee or in Hb A. How-ever, a similar problem exists with respect to the interpretationof all spectroscopic and crystallographic investigations ofstructure-function relationships in hemoglobin. Heme-hemeinteraction and resulting changes in the oxygen affinity of asubunit reflect differences in the ground-state free energies ofparticular conformational states of the hemoglobin molecule.Although these free energies undeniably depend upon theconformation, the connection between them is not readilyquantified. Spectroscopy tells us about the relations betweenground and excited states, and provides no direct informationabout the ground-state energies of various species relative toeach other. Likewise, our present knowledge of the potentialenergy of intramolecular interactions is so primitive that at-tempts to estimate changes in potential energy (as distinguishedfrom the free energy) at a chemically useful resolution (fractionsof kcal/mol) on the basis of changes in electron density mapsmust be regarded as speculative in the extreme. This is espe-cially so in a molecule whose structure and structural changesare as complex as those of hemoglobin. It must be thereforeadmitted that neither spectroscopic nor crystallographicmethods can provide direct information about heme-hemeinteraction that is free from doubt regarding its relevancy. Onthe other hand, it cannot be denied that measurements ofoxygen equilibrium are relevant, but the information providedby such measurements is macroscopic (thermodynamic) andtherefore not susceptible to any unique structural interpretation.

Both spectroscopic and crystallographic data have beenpresented in support of a concerted two-state description forthe oxygenation of hemoglobin (for recent reviews, see refs. 1

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and 42). The data are invariably interpreted on the assumptionthat certain spectral or structural changes associated withparticular alterations at an interface between subunits and/orin the vicinity of the heme groups are appropriate markers ordeterminants of changes in oxygen affinity at binding sites. Inthe absence of evidence to the contrary, it is equally valid toassume that the spectral changes reported here are likewisemarkers or determinants of changes in oxygen affinity at thebinding sites.

In conclusion, we believe that the results of this study con-stitute the most direct and least equivocal information on therole of protein structure in heme-heme interaction that has sofar been reported. We suggest that (i) these results are incom-patible with a two-structure model for heme-heme interactionand (ii) these results support the concept of direct ligand-linkedinteractions between subunits embodied in a sequential model.

We wish to thank Dr. A. V. Pisciotta for providing us with bloodsamples containing Hb M Milwaukee needed for our work. This workwas supported by research grants from the National Institutes of Health(HL-10383 and RR-00292) and the National Science Foundation (BMS72-02534). L.W.-M.F. is a recipient of the National Research ServiceAward (GM 05164-01).

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