Subunit Dissociation of Certain

Transcription

1 Subunt Dssocaton of Certan Abnormal Human Hemoglobns H. FRANKLIN BUNN From the Blood Transfuson Dvson, U. S. Army Medcal Research Laboratory, Fort Knox, Kentucky 4121 A B S T R A C T The extent of dssocaton of varous hemoglobns nto subunts was estmated from ther eluton volumes (V.) on G-1 Sephadex. Under the same controlled condtons carboxyhemoglobns A, A3 (A,), F, S, and C all had the same eluton volumes. The carboxy and cyanmet dervatves of hemoglobn Kansas (a varant wth very low oxygen affnty) had a relatvely hgh Ve, ndcatng a decreased mean molecular weght and therefore an ncreased tendency to form dmers and even monomers. Conversely, the lganded dervatves of hemoglobn Chesapeake (a varant wth hgh oxygen affnty) had a relatvely low V., suggestve of an mpared degree of subunt dssocaton. Deoxyhemoglobn Chesapeake had a V. dentcal wth that of deoxyhemoglobn A. Cat hemoglobn, known to have an unusually low oxygen affnty, was found to have a hgher V. than human, dog, rabbt, rat, or gunea pg hemoglobns. Haptoglobn s thought to bnd af dmers n preference to the a2,82-tetramer. The comparatve haptoglobn affntes of the human hemoglobns were measured by competton between the test hemoglobn and radoactve reference hemoglobn for haptoglobn bndng stes. Hemoglobns A, F, S, and C all seemed to bnd equally readly, but hemoglobn Kansas and cat hemoglobn showed a hgher affnty, and hemoglobn Chesapeake a lower affnty. These results are n accord wth recently proposed models whch predct that hemoglobns whch have an ncreased degree of subunt dssocaton wll have a low oxygen affnty, and vce versa. INTRODUCTION Most human hemoglobn varants generally have a sngle amno acd substtuton n ether the a- or pl-polypeptde chans. Such a structural alteraton wll com- Receved for publcaton 25 June 1968 and n revsed form 2 September monly confer a dfference n net surface charge allowng the abnormal proten to be detected and solated by electrophoretc and chromatographc methods. Many of these hemoglobns have no unusual chemcal or physcal propertes and appear to be functonally normal. A smaller number of human hemoglobn varants are assocated wth clncal features whch lead to ther detecton. These hemoglobns almost always have unusual propertes whch are readly demonstrable n vtro. A few human hemoglobn varants are charactered by marked abnormaltes n oxygen affnty (Table I). A mother and son wth cyanoss and a normal hematocrt were found by Ressman, Ruth, and Nomura to be heteroygous for a hemoglobn varant, desgnated as Kansas (1). The cyanoss was due to the low oxygen affnty of the whole blood. Recently Bonaventura and Rggs have determned the structure of solated hemoglobn Kansas and have confrmed that ths hemoglobn has a markedly reduced oxygen affnty and low hemeheme nteracton, but a normal Bohr effect (2). Fve hemoglobn varants: Chesapeake (3), Yakma (4, 5), Kempsey (6), and Raner (7) and Hroshma (8) have now been found to be assocated wth famlal erythrocytoss (Table I). In each nstance the affected ndvduals have been heteroygous for a hemoglobn wth a markedly hgh oxygen affnty. In addton, hemolysates contanng hemoglobn J-Cape Town have been found to have hgh oxygen affnty (9). However affected heteroygotes do not have erythrocytoss. The way n whch hemoglobn reversbly bnds oxygen has eluded precse understandng. Most pulng has been the mechansm by whch the oxygen affnty ncreases upon the stepwse addton of lgand: so called heme-heme nteracton. The conformaton of hemoglobn s known to be altered consderably upon oxygenaton (1). Furthermore, lgand bndng enhances the tendency of the tetramer to dssocate symmetrcally nto dmers: ac/t3 2a/3 (11, 12). These expermental observatons have formed the bass of a model proposed 126 The Journal of Clncal Investgaton Volume

2 TABL I Hemoglobn Varants Assocated wth Abnormal Oxygen Affnty Hemeheme Clncal features Oxygen nter- Hemoglobn varant n heteroygote affnty acton Kansas a22l12 Thr Cyanoss Low Low Normal hct Chesapeake 2I92 LeuB2 Yakma a Hs Kempsey a Asn rythro- Hgh Low Raner a Hs cytoss Hroshma a Aspj J-Cape Town a292 Gln132 Normal hct Hgh? Thr, threonne; Leu, leucne; Hs, hstdne; Asn, asparagne; Asp, asparatc acd; Gln, glutamne. by Benesch, Benesch, and MacDuff for the reacton of lgands wth hemoglobn whch postulates that the a,8 dmer s the prmary functonng unt (13). Stepwse oxygenaton s thought to nvolve rapd exchange between oxygenated and deoxygenated dmers. It s of great nterest to examne those hemoglobns wth abnormal lgand affnty n the lght of ths model. It predcts that f a hemoglobn varant dssocates more readly than normal from a lganded tetramer to dmer t wll have a low oxygen affnty, and vce versa. It s noteworthy that the amno acd substtuton n all of the hemoglobns shown n Table I except Raner and Hroshma s n the nonhelcal FG segment, or adjacent G helx, regons whch are n the nterface between adjonng aft-dmers (14). The substtuton n hemoglobns Raner and Hroshma s n the H helx whch s contguous wth the FG segment. A structural alteraton n these areas mght well effect a marked change n the equlbrum between dmer and tetramer. In these experments the extent of subunt dssocaton of varous normal and abnormal human and anmal hemoglobns was studed by measurng ther relatve eluton volumes on G-1 Sephadex, a cross-lnked dextran whch serves as a molecular seve. In addton, the relatve haptoglobn bndng of these hemoglobns was tested. MTHODS Preparaton of hemoglobn samples. Blood specmens were collected n acd ctrate dextrose (ACD). Hemolysates were prepared from blood specmens as prevously descrbed (15), and unless stated otherwse, were gassed wth carbon monoxde and stored at 4VC. Hemolysates were dalysed aganst the approprate elutng buffer before hemoglobn purfcaton by column chromatography. Isolated hemoglobns were concentrated by ultrafltraton and agan gassed wth carbon monoxde. Blood specmens from an ndvdual heteroygous for hemoglobn Chesapeake were maled by Ar xpress. Hemoglobn Chesapeake was separated from hemoglobn A by chromatography on carboxymethylcellulose as descrbed by Charache, Weatherall, and Clegg (3). xperments on hemoglobn Chesapeake were performed wthn 1 wk after blood samples were obtaned. Hemoglobns Kansas and A were solated from the hemolysate of a heteroygote by chromatography on carboxymethylcellulose by Drs. Joseph Bonaventura and Austen Rggs (2) and ar-shpped n the carboxy form packed n ce. In lke manner hemoglobns Yakma and A from a heteroygote were solated by chromatography on dethylamnoethyl (DA)-Sephadex by Dr. Demetros Rgas (4) and ar-shpped. Hemoglobn F was solated from a fresh sample of cord blood on Amberlte (CG-5) by the method of Allen, Schroeder, and Balog wth the use of developer 2 (16). Wth ths procedure hemoglobn A3 (A1)1 was solated from a hemolysate of a normal adult. Hemoglobns C and S were solated from the blood of a patent wth hemoglobn SC dsease by chromatography on carboxymethylcellulose (17). Hemoglobn S from the hemolysate of a homoygote was also tested wthout further purfcaton. In these experments hemoglobns-59fe were used as markers. Rabbt hemoglobn was labeled as prevously descrbed (18). Human hemoglobn A-'Fe was prepared by the exchange of heme between unlabeled ferrhemoglobn A and 'Fe-labeled methemalbumn. Ths method, descrbed n detal elsewhere (15), allowed much hgher specfc actvty than s possble by the ncubaton of human retculocytes wth 5'Fe. In bref hemn-55fe was crystalled from rabbt hemoglobn-"5fe and was then bound to human serum albumn.2 A mxture contanng methemalbumn-'fe and a heme equvalent amount of unlabeled ferrhemoglobn A was ncubated for 1 mn at 37 C and then dalyed aganst developer 2 at 4 C. Under the expermental condtons employed there was no net loss of heme from the hemoglobn. The labeled cyanmethemoglobn A was separated from the methemalbumn-5fe on Amberlte CG-5 (16). luton volumes of hemoglobns on G-1 Sephadex. A column of G-1 Sephadex 3 measurng 95.5 X 2.4 cm was used n all these experments. When oxy- or carboxyhemoglobns were studed the column was equlbrated wth a soluton consstng of two parts sotonc NaCl and one part sotonc phosphate buffer, ph 7.4 (buffered salne). Durng the chromatograms of carboxyhemoglobn, t was not practcal to keep the buffer saturated wth carbon monoxde, so that some of the lgand may have been lost or replaced by oxygen. When ferrhemoglobn cyande was tested, the buffer contaned cyande (2 mmoles/lter). When deoxyhemoglobn was appled to the column the buffer contaned sodum dthonte (1.5 mmoles/lter), and 1%o ntrogen was bubbled slowly nto the buffer n the supply bottle to exclude as much oxygen as possble from the system. Most of the experments were done at room temperature. Before runnng the column at 4 C, t was repacked to ts orgnal dmensons n the cold. Samples of 1.5 ml were appled to the column and unless stated otherwse conssted of 8 mg/ml of hemoglobn n buffered salne. In some experments human albumn labeled wth I 4 served as a 1 A3 or A1 hemoglobn refers to the mnor fractons whch are more electronegatve than hemoglogn A and comprse about 8% of the adult human hemolysate. 2 Nutrtonal Bochemcals Corporaton, Cleveland, Oho. 3 Pharmaca Fne Chemcals Inc., Pscataway, N. J. 4 Squbb Rado-odnated ("li) Human Serum Albumn USP. Hemoglobn Subunt Dssocaton 127

3 marker. 1.5 mg of albumn-=i, havng an actvty of.1-.5,uc, was mxed wth the hemoglobn soluton mmedately before applcaton. A flow rate of 2-3 ml/hr was mantaned. Fractons of equal volume (3.62 ml) were obtaned by a Glson lnear fractonator (Model VL). The hemoglobn concentraton of successve fractons was obtaned from the absorbance of an approprate dluton n Drabkns soluton, measured at 54 m,& or, wth more dlute solutons, at 415 mu. Radoactvty of an alquot of successve fractons was measured n a well scntllaton counter. The relatve Sephadex moblty of a gven substance can be expressed by the constant Kav whch s ndependent of the column dmensons and the extent of packng (19). K5V = Ve~-VO Vt - VI' The eluton volume V. of a substance was determned by the volume of effluent between ts pont of applcaton and ts peak concentraton durng eluton. The vod volume Vo was determned by the eluton volume of Dextran Blue,3 a hgh molecular weght polymer whch s entrely excluded by the gel matrx and therefore has maxmal moblty. Vt was the total bed volume of the column. The determnaton of V. for hemoglobn was subj ect to expermental error such as slght varablty n the ntaton of collecton and n the fracton volumes. Ths uncertanty could be mnmed by the use of albumn-"'1i as a marker. Chromatography of a mxture of hemoglobn and albumn-=i allowed sharp resoluton of the two components despte some degree of overlap snce ther respectve concentratons were measured by dfferent means. In the data reported n Table II and Fg. 1, mxtures contanng the test hemoglobn and a small amount of albumn-'i were chromatographed. The V. of hemoglobn was corrected for slght varatons n the V. of the albumn-mi. The resoluton of hemoglobns on G-1 Sephadex was greatly ncreased by chromatographng a mxture contanng equal amounts of the test hemoglobn and a '9Fe-labeled reference hemoglobn. In these experments the appled samples contaned a total of 8 mg/ml of hemoglobn n ether the carboxy or cyanmet form and were chromatographed at 25C, snce under these condtons subunt dssocaton appeared to be enhanced (see Results). The specfc actvty of a gven fracton SAf (cpm/mg) was calculated from ts radoactvty Af (cpm/ml) and ts hemoglobn concentraton Cf (mg/ml). SAf SAf = Cf, Cf The concentraton of the labeled reference hemoglobn Cr and that of the unlabeled test hemoglobn Ct n each fracton was calculated as follows: Cr Af SAo where SAo s hemoglobn. the specfc actvty of the orgnal labeled Ct = Cf - Cr. In these and other experments, the hemoglobn A solated from the same hemolysate as the abnormal hemoglobn was always tested for drect comparson. Measurement of haptoglobn bndng. The relatve bndng affntes of varous human hemoglobns to haptoglobn were tested by a competton approach descrbed n detal elsewhere (18). In bref, a mxture of equal amounts of the carboxy dervatves of unlabeled test hemoglobn and rabbt hemoglobn-'fe was added n excess to serum of known haptoglobn phenotype. The bound hemoglobn was then separated wth G-1 Sephadex. The bndng of the test hemoglobn relatve to the reference labeled hemoglobn was calculated as follows: % Hp bound by test Hb = 1 _ 5 SAHVII, SAmxture Mscellaneous procedures. Carboxyhemoglobn was converted nto oxyhemoglobn as recommended by Rggs and TABL I I luton Data for Hemoglobns and Other Substances on G-1 Sephadex Number of Concentratons of Ve alb Substance determnatons appled sample Temperature V. Ksv mg ml C Dextran blue Human albumn (1.).224 Oxyhemoglobn A Oxyhemoglobn A ( )*.353 Oxyhemoglobn A Carboxyhemoglobn A Cyanmethemoglobn A Deoxyhemoglobn A ( )*.338 Cyanmethemoglobn Chesapeake Cyanmethemoglobn Kansas Dextran blue Human albumn (1.).225 Oxyhemoglobn A ( )*.348 Oxyhemoglobn A * Mean and range. t stmated from data n Fg H. F. Bunn

4 Herner (2). Cyanmethemoglobn was prepared from oxyhemoglobn as prevously descrbed (15). Methemoglobn was determned by the method of velyn and Malloy (21). Alkal denaturaton was tested by the procedure devsed by Husman and Meyerng (17). One part of 12 N NaOH was added to 1 parts of oxyhemoglobn dssolved n.1 M phosphate buffer, ph The fnal ph of ths mxture was Under these condtons the rate of denaturaton of hemoglobn A was slow enough (half-tme 3 mn) to detect subtle changes n other hemoglobns wth whch hemoglobn A was compared. Auto-oxdaton of oxyhemoglobn Chesapeake and A (3 mg/ml) was measured at 37C n.1 M phosphate buffers at ph 6.6 and 7.4. RSULTS xperments were frst done to confrm that the K., of hemoglobn on G-1 Sephadex was useful as an ndcator of the extent of subunt dssocaton. Condtons whch enhance the subunt dssocaton of hemoglobn wll result n a lower mean statstcal molecular-weght and therefore an ncrease n K.,. Conversely, a decreased K., would be expected n any hemoglobn whch has an mpared degree of dssocaton. Human albumn was found to have a consderably lower eluton volume (and therefore a lower K.y) than all the hemoglobns tested (Table II), n agreement wth the fndngs of Andrews (22). Only part of ths dfference could be due to the slght dfference n molecular weght between human albumn and hemoglobn (69, vs. 64,5). The eluton volume of hemoglobn would be further ncreased by the extent to whch t dssocates nto dmers durng the chromatography. Oxyhemoglobn, cyanmethemoglobn, and carboxyhemoglobn all had very smlar Kavs (Table II). Gudott, usng osmotc pressure measurements, found that the frst two forms had dentcal degrees of subunt dssocaton, but that of carboxyhemoglobn was somewhat less (12). In contrast, the eluton volume of deoxyhemoglobn was consstently less than that of the lganded forms (Fg. 1, Table II). It was not techncally possble to run the column under the strctly anaerobc condtons necessary to mantan hemoglobn n ts deoxygenated form. Therefore the deoxyhemoglobn was chromatographed n the presence of dthonte. There s evdence that ths reducng agent may cause certan denaturatve effects whch could alter ts physcal and chemcal propertes (23). Nevertheless, the-relatvely low Kav of deoxyhemoglobn suggests a lower degree of subunt dssocaton n agreement wth the gel fltraton data of Merrett (24), the sedmentaton data of Benesch and assocates (11), and the osmotc pressure measurements of Gudott (12). luton volumes of oxyhemoglobn were measured over a wde concentraton range. Mass acton law appled to self-assocatng systems would dctate that dlute solutons have a greater degree of subunt dssocaton than concentrated ones. The Kav of oxyhemoglobn was found to vary nversely wth ts concentraton (Table II) n 1 deoxy hemoglobn I oxy hemoglobn - o5 o - I. U l( 5( albumn-- vod! volume I /.' 5 55 FRACTION -No ~ mu & FIGUR 1 luton profles of oxyhemoglobn A, deoxyhemoglobn A, and human serum albumn on G-1 Sephadex. Chromatograms of oxyhemoglobn (---) and deoxyhemoglobn (-) were each run n trplcate. The resoluton of these hemoglobns was enhanced by chromatographng a mxture contanng the hemoglobn and a small amount of human albumn-'l. The eluton profle of the albumn (- - -) was determned from radoactvty of successve fractons. Hemoglobn Subunt Dssocaton 129

5 .4- -q.3- l v,2- u.1-.a 1-.5 O *,4 -,3 9 Z',21.A.11 I % A I % / jkansas /~~~~~~~~1 I~~~~~1 m - -- a * * m 2-,, a 4(% 15- v 1- _V 'U 5- I~ I I I *-.%/ a\\ SPCIFIC ACTIVITY ~~~~~~~~~~~~~~I I Ir I Itt yi m Iff --.: I I8 8 5 FRACTION I I I I No. FIGUR 2 luton profle of cyanmethemoglobn Kansas and cyanmethemoglobn A-'Fe. A mxture contanng equal amounts of the two hemoglobns was run on the G-1 Sephadex column. From -6 the optcal densty and radoactvty measurements of successve fractons (lower panel), the specfc actvtes of these fractons were calculated (mddle panel). The 4 top panel shows the resoluton of the two ' hemoglobns, as calculated from the expermental data (see Results). The small -2 peak under the hemoglobn A W peak (---) represents unlabeled hemoglobn of unknown dentty and queston- _ able sgnfcance. 9 agreement wth the data of Andrews (22) and Gudott (25). Concentraton dependence of hemoglobn dssocaton has also been found from osmotc pressure measurements (12) and n recent sedmentaton data (26). The eluton volumes of oxyhemoglobn solutons were greater at 25 than 4VC. Ths change s unlkely to be due to a sgnfcant alteraton n the dstrbuton of water nsde and outsde the gel matrx, snce the moblty of albumn was unaffected by the temperature change (Table II). Obrnk, Laurent, and Rgler have shown that the Kay of Fcoll fractons on G-2 Sephadex vared only slghtly over a wde temperature range (9-6'C) (27). Our results suggest that the subunt dssocaton equlbrum of oxyhemoglobn ncreases slghtly wth temperature. Benesch and assocates have some ndrect expermental evdence supportng ths concluson (13). However Krschner and Tanford found that the sedmentaton of human carboxyhemoglobn was ndependent of temperature (28). The eluton volumes of varous abnormal human hemoglobns were compared. Chromatography of a mxture of labeled reference hemoglobn allowed much greater resoluton than was possble by measurement of the Ka. of the test hemoglobn alone. If the test hemoglobn has the same degree of subunt dssocaton as the reference hemoglobn, then they should have dentcal eluton volumes on G-1 Sephadex, and the specfc actvtes of successve fractons should be dentcal. Ths was found to be true for carboxyhemoglobns A3, F, S, and C. The on-exchange capacty of G-1 Sephadex appeared to be nsgnfcant snce these hemoglobns have wdely dfferent surface charges and yet had eluton volumes dentcal wth carboxyhemoglobn A-2Fe, and therefore to each other. When carboxyhemoglobn Kansas was tested, a marked fall n specfc actvty of successve fractons ndcated that the unlabeled test hemoglobn had a greater eluton volume than the reference hemoglobn, and therefore a lower mean statstcal molecular weght ndcatve of an ncreased degree of subunt dssocaton. Identcal results were obtaned when the cyanmet dervatves of hemoglobn Kansas and A-"9Fe were tested (Fg. 2). The K., of Kansas was 13 H. F. Bunn

6 U a_.3- U "I*- Z.2- A.1 M > U 15-49I., _ f 1' v L" 5. U. m $A oi. A Kansas SPCIFIC ACTIVITY 6 I-. Z- Lu Z a O A x I FRACTION No A 2 c FIGUR 3 luton profle of cyanmethemoglobn A, solated from the Kansas hemolysate, and cyanmethemoglobn A-'Fe. hgher than that of any of the other hemoglobns tested (Table II). When the most dlute soluton of hemoglobn A was chromatographed, ts ntal concentraton at the pont of applcaton was 1.6 mg/ml, and ts mean concentraton upon eluton was.73 mg/ml. Under these condtons, durng the run most of the hemoglobn would be expected to be n the form of dmers, as estmated from recently derved equlbrum constants (12, 28, 29). The fact that the eluton volume of hemoglobn Kansas was far greater suggests that dssocaton had extended to the formaton of monomers. These fndngs are n agreement wth the sedmentaton velocty data of Bonaventura and Rggs (2). They found that carboxy- and oxy- but not deoxyhemoglobn Kansas had low sedmentaton coeffcents ndcatng that Kansas dssocated nto dmers at concentratons n whch hemoglobn A remaned as an ntact tetramer. The abnormal Sephadex moblty of hemoglobn Kansas was not lkely to be due to any alteratons resultng from ts purfcaton snce the hemoglobn A solated from the same column showed an eluton volume dentcal wth the A-'Fe reference hemoglobn (Fg. 3). Hemoglobn Chesapeake had a lower eluton volume than the labeled hemoglobn A (Fg. 4). Both the carboxy- and ferrhemoglobn cyande dervatves gave dentcal and reproducble results on a total of sx chromatograms prepared from hemoglobns solated from two separate blood samples. Hemoglobn A solated on the same preparatve column as the Chesapeake A was also tested each tme and found to have an eluton volume dentcal wth the A-Fe reference hemoglobn (Fg. 5). When cyanmethemoglobn Chesapeake was run alone wth the albumn-'i marker, ts K.y was not very much less than that of cyanmethemoglobn A obtaned from the same column (Table II). Ths small dfference s probably a reflecton of the much hgher resoluton offered. by the chromatography of hemoglobn mxtures. In contrast to the carboxy and cyanmet dervatves, deoxyhemoglobn Chesapeake showed an eluton volume dentcal wth that of deoxyhemoglobn A-w'Fe. The results from ths experment very closely resembled those shown n Fgs. 3 and 5. Fnally when a mxture of the cyanmet dervatves of Chesapeake and A-Fe was run at 4VC, only a slght ncrease n specfc actvty of Hemoglobn Subunt Dssocaton 131

7 The eluton volumes of varous anmal hemoglobns were measured by chromatographng a mxture of the anmal hemoglobn and human hemoglobn A-mFe, under the same condtons employed n the prevous experments (Fgs. 2-5). The cyanmet and carboxy dervatves gave smlar results. Dog and gunea pg hemoglobns showed eluton volumes dentcal wth human. The results for these experments were very smlar to the data shown n Fgs. 3 and 5. Cat hemoglobn had a larger eluton volume than human hemoglobn (Fg. 6). The eluton volumes of rabbt and rat hemoglobns were slghtly greater than the human, dog, and gunea pg hemoglobns but less than the cat hemoglobn. g.3 mu - U ] A / 3- v- u - a successve fractons was noted ndcatng less of a dfference n eluton volumes than at 25C. Perhaps ths s because subunt dssocaton of hemoglobn A appears to be reduced at the lower temperature (Table II). These results ndcate that the lganded but not the deoxy form of hemoglobn Chesapeake have decreased eluton volumes and therefore a reduced degree of subunt dssocaton when compared to hemoglobn A. Under the same condtons used for hemoglobn Chesapeake (Fg. 4), no dfference between the eluton volume of hemoglobn Yakma (and ts companon A hemoglobn) and hemoglobn A-Fe could be demonstrated. 2- I5-1- loo - SPCIFIC ACTIVITY U.5' ud -- #A 3-.4' 'u t u u _% C. O 8.3 ' 4 2,2 FIGUR 4 FRACTION No. luton profle of cyanmethemoglobn Chesapeake and cyanmethemoglobn A-"Fe. 132 H. F. Bunn

8 6- - CD Ad -o I 1 Chesapeake > 2- _ u 15- _ WI loo- / SPCIFIC ACTIVITY X, M- 5- I I I I I I.5 - O.4' 6- a' Ix 1.3' Z - Wu Z.2 O -I x.1' 6 4.: 2 FIGuR 5 The relatve affntes of the varous human hemoglobns for haptoglobn are shown n Fg. 7. If the test hemoglobn had precsely the same affnty as the rabbt hemoglobn-'fe, then the specfc actvty of the solated Hb-Hp complex would be dentcal wth that of the orgnal hemoglobn mxture. xactly 5% of the haptoglobn would be bound by the test hemoglobn. In fact most of the test hemoglobns showed a slghtly greater affnty than the labeled rabbt hemoglobn, formng complexes wth about 52-55% of the haptoglobn. These results agree well wth earler data on hemoglobn A (18). In contrast to hemoglobns A, F, S, and C, hemoglobn Kansas showed a consderably greater affnty for haptoglobn, whereas hemoglobns Chesa FRACTION No. luton profle of cyanmethemoglobn A, solated from the Chesapeake hemolysate, and cyanmethemoglobn A-'Fe. peake and Yakma showed somewhat decreased affnty. Haptoglobn phenotype had no effect on ts bndng to the varous test hemoglobns. In a parallel experment, a mxture contanng equal amounts of unlabeled cat hemoglobn and 'Fe-labeled human hemoglobn was added n excess to human serum (type 1-1 haptoglobn). 62% of the haptoglobn complexed wth the cat hemoglobn, 38% wth the human. Oxyhemoglobn Chesapeake and ts companon oxyhemoglobn A (solated from the same on-exchange column) both showed equal rates of alkal denaturaton. Furthermore these hemoglobns both auto-oxded at the same rate. Hemoglobn Subunt Dssocaton 133 *

9 49 UA v.a.11 3o I- 1-4 u U am IL SPCIFIC ACTIVITY.5*.8 a.4 - Q.2 *6 4.S 2 s I ' FIGUR 6 FRACTION luton profle of the cyanmet dervatve of cat hemoglobn and cyanmethemoglobn A-'Fe. No. 134 H. F. Bunn

10 % OF Hp BOUND BY TST HMOGLOBIN I I A A (Kansas Column) A (Chesapeake Column) A (Yakma Column) * Co C Kansaso Chesapeake Yakma -IL O =1-1 Hp FIGUR 7 Relatve haptoglobn bndng of varous human hemoglobns. To a mxture contanng equal amounts of carboxy-9fe rabbt hemoglobn and the carboxy hemoglobn to be tested was added a small amount of normal serum, of haptoglobn phenotype 1-1 (), or 2-2 (). The per cent of the haptoglobn bound to the test hemoglobn was calculated from the specfc actvty of the solated hemoglobn-haptoglobn complex (see Results). *=2-2 Hp DISCUSSION One of the long-standng nterests n hemoglobn chemstry has been the dssocaton of the molecule nto smaller fragments. Subunt formaton was readly seen after subjectng hemoglobn to varous stresses such as extremes of ph or very hgh onc strength (3). As the proten structure was elaborated t appeared lkely that subunt dssocaton nvolved dsrupton of lnkages between the component a and polypeptde chans. Vnograd and Hutchnson presented convncng evdence that the tetramer splt symmetrcally (31): a28t2 2a,8. xperments along several dfferent lnes ndcated that ths equlbrum exsted under physologc condtons of ph and onc strength (12, 13, 32) and therefore mght be an mportant determnant of hemoglobn functon. Subunt dssocaton has been approached by a varety of methods, all of whch nvolve the measurement of average molecular weght. The observed molecular weght can be related to that of the ntact molecule and serves as an ndex of the extent of dssocaton. Osmotc pressure measurements (12) and sedmentaton velocty data (28) have been used to calculate an equlbrum constant for hemoglobn dssocaton under physologc condtons. Ackers and Thompson (29), and more recently Chancone, Glbert, Glbert, and Kellett (33), have used G-1 Sephadex to study hemoglobn dssocaton and have calculated equlbrum dssocaton constants for human oxyhemoglobn. In order to obtan these quanttatve results t was necessary to load the column wth a large enough volume of hemoglobn soluton to acheve a plateau type eluton profle. In the experments reported here and n those of Andrews (22), small volumes were appled, resultng n the formaton of sharp peaks. Although ths expermental approach provded qualtatve data, t does not permt the calculaton of equlbrum constants. However, the data shown n Table II confrm that t can be useful, snce condtons known to affect hemoglobn dssocaton are reflected n expected alteratons n hemoglobn moblty. In these experments the eluton volume of sngle hemoglobn solutons was not senstve enough to detect subtle changes n subunt dssocaton. When lganded forms of hemoglobn Chesapeake were run n a mxture wth an equal amount of hemoglobn A-69Fe, a consstent rse n specfc actvty of successve fractons ndcated that the Chesapeake had a lower eluton volume and therefore a decreased tendency to dssocate nto dmers. xchange of ntact heme groups, a phenomenon whch proceeds qute readly n mxtures contanng methemoglobn, was very unlkely to affect the results cted above. Carboxy and cyanmet dervatves were run at 25C, condtons under whch heme exchange does not occur (15). However, another source of error may be Gudott has osmotc pressure data n- more sgnfcant. dcatng that n mxtures of two dfferent hemoglobns, say (a2,2) and (a''a2), the hybrd afa'#' escapes detecton by any method dependent on charge dfferences for resoluton (34). In the chromatographc runs contanng mxtures of hemoglobn A and Chesapeake, the extent that Hemoglobn Subunt Dssocaton 135

11 the hybrd aaach-fa was present would attenuate the resoluton of the two parent molecules nto separate peaks. The extent of ths phenomenon cannot be measured drectly, but t was unlkely to be a major source of error snce there was lttle dfference between the Sephadex moblty of hemoglobn Chesapeake when measured alone (Table II) as compared to ts moblty n a mxture wth the labeled reference hemoglobn (Fg. 4). The falure to observe a dfference between the eluton volumes of hemoglobn Yakma and the reference hemoglobn does not support the contenton that Yakma lke Chesapeake has a decreased tendency to dssocate. Ths possblty s not entrely ruled out, however. The extent of formaton of the nterspeces hybrd (a2/sai3yakma) may have been great enough to preclude resoluton of the two hemoglobns. It was pertnent to compare the affntes of the varous hemoglobns to haptoglobn. Ths plasma proten bnds specfcally and rreversbly to hemoglobn. 1 mole of type 1-1 haptoglobn s known to combne stochometrcally wth 1 mole of hemoglobn (35). Snce type 1-1 haptoglobn s a symmetrcal molecule (36), t s lkely to have two bndng stes. vdence from several dfferent expermental approaches ndcates that haptoglobn bnds separate a,# dmers rather than the ntact hemoglobn tetramer (18, 37-39). If so, a hemoglobn whch has an ncreased tendency to dssocate nto half molecules would have a relatvely hgh affnty for haptoglobn, and vce versa. When vewed n ths way, the relatvely hgh haptoglobn affnty of hemoglobn Kansas and cat hemoglobn and the low haptoglobn affnty of hemoglobn Chesapeake are n good agreement wth the Sephadex moblty data. However, t s also possble that these hemoglobns have alteratons n tertary structure whch affect ther affntes for haptoglobn. Comparable data for hemoglobn A treated wth varous sulfhydryl agents offer an nterestng comparson (18). P-mercurbenoate-treated hemoglobn, known to have an enhanced degree of dssocaton nto subunts, had a relatvely hgh haptoglobn affnty. Conversely hemoglobn reacted wth bs- (N-malemdomethyl) ether (BM) has a markedly mpared degree of dssocaton (4) and a low affnty for haptoglobn. The G-1 Sephadex moblty of BM hemoglobn was consderably greater than that of untreated hemoglobn-'9fe wth whch t had been mxed,5 a fndng smlar to that obtaned for hemoglobn Chesapeake (Fg. 4). The smlartes between hemoglobn Chesapeake and BM hemoglobn extend even further. Of all the sulfhydryl reagents, BM appears to effect the greatest ncrease n oxygen affnty (4-42). Benesch and assocates have proposed a model (13) whch s based on two mportant propertes of hemo- 5Unpublshed observaton. 136 H. F. Bunn globn: ts change n conformaton upon reacton wth lgand and ts reversble dssocaton nto dmers. More recently Gudott has derved a comprehensve seres of equatons whch relate subunt dssocaton and lgand bndng n quanttatve terms (43). When hemoglobn concentraton s large relatve to the values of the dssocaton equlbrum constants, a condton certanly exstng n the red cell, the followng smplfed equaton can be wrtten (43): p( Ko \1/4 P-\K31 Kd, where Pso s the partal pressure at whch half of the hemoglobn s bound by lgand (such as oxygen); Ko and Kdeo are the dssocaton equlbrum constants for the oxygenated and deoxygenated tetramers respectvely, and Ks s the assocaton equlbrum constant for the reacton of the af dmer wth oxygen. It can be readly apprecated from ths equaton that f the rato of the dssocaton constant of lganded hemoglobn over that of unlganded hemoglobn s abnormally hgh, as s true for hemoglobn Kansas, there wll be an ncrease n Po, or a decrease n oxygen affnty. It s of nterest to apply the equlbrum constant derved by Bonaventura and Rggs for oxyhemoglobn Kansas to ths equaton. Rggs' and Bonaventura's sedmentaton data ndcate that KdeoKans = KdeoA. If K3Kans=K3A, an assumpton that as yet has no expermental bass, then, from expermental values for KOA ( X 1-' mole/lter [44, 45]) and KoKans (2 X 1-" [2]), the followng rato can be calculated: P5A/P5K..S =.28. The rato P3oA/P5Kan. derved from the oxygen dssocaton curves of Rggs and Bonaventura s.3 (2). Ths good agreement lends support for the models proposed by Benesch et al. and by Gudott. Further support s provded by a comparson of oxygen affnty and subunt dssocaton of certan anmal hemoglobns. Cat hemoglobn, for example, has a much lower oxygen affnty than that of other mammals (46). arly sedmentaton data suggested that cat hemoglobn dssocated readly n dlute soluton, although the data are dffcult to nterpret because of varablty n expermental condtons (47). Sullvan recently reported that cat hemoglobn eluted after human hemoglobn on G-1 Sephadex but presented no data (48). The results shown on Fg. 6 confrm that cat hemoglobn has a large eluton volume and therefore an ncreased degree of subunt dssocaton. The adult heteroygous sheep has two hemoglobns, types A and B, whch are readly separated by electrophoress. The "slow" hemoglobn, type B, has a much lower oxygen affnty than the "fast" type A hemoglobn (49). Glbert, usng a hghly senstve gel fltraton approach, smlar n prncple to the one used n the present studes, has recently shown that type B sheep hemoglobn dssocates more readly than type A (5).

12 The abnormally hgh oxygen affnty seen n certan hemoglobn varants (Table I) may be due to a low degree of subunt dssocaton of the lganded form. The data on hemoglobn Chesapeake whch have been presented here suggest ths explanaton, but other factors may also be contrbutory. It may be that the amno acd substtuton n Chesapeake (and smlar varants) confers subtle conformatonal changes whch per se could alter lgand affnty. Nagel, Gbson, and Charache have shown that deoxyhemoglobn Chesapeake dffers from deoxyhemoglobn A n reactvty to odoacetamde and the onaton of tyrosne resdues (51). In contrast only the lganded forms of the two hemoglobns dffer n ther Sephadex eluton volumes. Furthermore, Nagel and colleagues showed that carbon monoxde reacted wth Chesapeake twce as rapdly as A, but ths observaton does not exclude the postulated mportance of subunt dssocaton as a determnant of lgand affnty. Hemoglobn Chesapeake had a normal rate of alkal denaturaton. It s noteworthy that hemoglobn Raner, whch resembles Chesapeake n ts clncal features and functonal propertes (Table I), s the frst adult human hemoglobn found to be alkal resstant (7). Ths phenomenon whch s so strkng n hemoglobn F s not lkely to be due to a decreased tendency to form dmers, snce hemoglobn F appears to have a normal moblty on G-1 Sephadex. Furthermore, hemoglobn Chesapeake and BM hemoglobn,8 both of whch appear to dssocate less readly nto half molecules, have rates of alkal denaturaton smlar to hemoglobn A. Fnally the very hgh ph at whch denaturaton s measured would be expected to favor dmer formaton n all hemoglobns. What seems more plausble s that resstance to alkal denaturaton s due to decreased dssocaton of dmer to monomer. Hemoglobn F forms nterspeces hybrds much less readly than hemoglobn A (52), a phenomenon dependent upon the formaton of monomer. Furthermore, hemoglobn Kansas has a greater rate of alkal denaturaton than hemoglobn A (2). The data shown n Table II and Fg. 2 suggest that Kansas has an ncreased tendency to form monomers as well as dmers. All the hemoglobn varants depcted n Table I have a low n value, ndcatve of decreased "heme-heme" nteracton. Ths fact s not predctable from Gudott's model merely on the bass of alteratons n the extent of subunt dssocaton and pont to conformatonal changes n the Oa-dmers themselves, whch would affect cooperatve nteractons. The fndngs of Nagel and coworkers (51) ndcate that ths s probably true for hemoglobn Chesapeake. Furthermore, the apparent tendency for hemoglobn Kansas to form monomers may underle a decrease n cooperatve nteractons. 6 Unpublshed observaton. ACKNOWLDGMNTS Drs. Samuel Charache, Demetros Rgas, and Austen Rggs kndly furnshed the blood and hemoglobn specmens whch were so essental for ths study. I am especally ndebted to Dr. Rggs for hs constructve comments and for revewng ths manuscrpt. Dr. Gerald Bloom performed the haptoglobn typng. RFRNCS 1. Ressmann, K. 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