The Modification of Hemoglobin by Citrate*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The Americn Society for Biochemistry nd Moleculr Biology, Inc. Vol. 268, No. 21, Issue of July 25, pp , 1993 Printed in U.S.A. The Modifiction of Hemoglobin by Citrte* (Received for publiction, December 14, 1992, nd in revised form, Februry 19, 1993) Peter J. Anderson From the Deprtment of Biochemistry, University of Ottw, Ottw, Ontrio K1H 8M5, Cnd Citrte, when ctivted by wter-soluble crbodiimide, covlently modifies hemoglobin. At ph vlues ner neutrlity, complete modifiction of the N-terminI vline residues of - chins cn be ccomplished with high degree of specificity. These groups rect t much more rpid rte thn slower recting set of functionl groups. Modifiction of hemoglobin with citrte lters the oxygen ffinity of the protein. Although the p50 is not chnged, the coopertive nture of oxygen binding is gretly decresed. Hemoglobin S modified with citrte is more soluble thn unmodified hemogiobin S. The time tken for deoxygented hemoglobin S to come out of solution in concentrted phosphte solutions is incresed by citrte modifiction. Interest in cell-free blood substitute bsed on hemoglobin hs led to the exmintion of chemicl mens of modifying hemoglobin structure to mke it useful for oxygen trnsport nd delivery to tissues. Modifiction is needed becuse outside the red blood cell the ioniclly bound llosteric effector 2,3- bisphosphoglycerte (BPG) dissocites nd is lost during purifiction of hemoglobin resulting in n uncceptbly high ffinity of the purified protein for oxygen. In ddition, concentrtions of hemoglobin which cn, becuse of osmotic considertions, be infused re sufficiently dilute tht dissocition to dimers occurs to the extent tht there is rpid renl clernce resulting in high rte of removl of infused hemoglobin from circultion nd possible renl dmge. This leds to the need for chemicl cross-linking of infused hemoglobin which is to be used s blood substitute. Bifunctionl nionic regents ble to rect with mino groups which would Chemicl Co. nd crried out t 0 C in n ice bth. [ CICitrte (Du be expected to bind with high ffinity t the BPG binding site Pont-New Englnd Nucler) ws used to determine the extent nd nd both decrese the oxygen ffinity nd provide covlent cross-link preventing dimer formtion hve received prticulr ttention recently (Fntl et l., 1987; Kvnugh et l., 1988; Benesch nd Kwong, 1988; Vndegriff et l., 1991; Berbers et l., 1991). Another re where chemicl modifiction of hemoglobin could be of clinicl benefit is the modifiction of hemoglobin S. The use of vriety of regents which rect with protein mino groups hs been described. These hve been used with view to preventing polymeriztion of the deoxy form of hemoglobin S, which leds to the sickling *This workws supported in prt by grnt from the Cutter Biologicls/Cndin Red Cross Reserch nd Development Fund. The csh of publiction of this rticle were defryed in prt by the pyment of pge chrges. This rticle must therefore be hereby mrked dvertisement in ccordnce with 18 U.S.C. Section 1734 solely to indicte this fct. The bbrevitions used re: BPG, 2,3-bisphosphoglycerte; MOPS, 4-morpholinepropnesulfonic cid; EDC, l-ethyl-3-(3-dimethylminopropy1)crbodiimide; HPLC, high performnce liquid chromtogrphy; FPLC, fst protein liquid chromtogrphy; Hg, hemoglobin; OPA, o-phthlldehyde of red blood cells (Klotz et l., 1981, 1985; Chtterjee et l., 1986). Hemoglobin contins region in which there is concentrtion of positive chrges from imidzoles nd mino groups tht form the BPG binding site (Perutz et /., 1980). A vriety of liphtic nd romtic poly(crboxy1ic cids) hve been shown to interct wit hemoglobin to reduce oxygen ffinity, presumbly due to electrosttic binding to this region of the molecule (Shimizu nd Bucci, 1974). Since poly(crboxy1ic cids) contin more thn one rective group tht cn be ctivted by crbodiimides to rect with nucleo- philic mino groups (Hore nd Koshlnd, 1967), it is possible tht the use of ctivted crboxyl groups could provide mens of covlently modifying nd perhps cross-linking hemoglobin. Chemicl modifiction of this type could be of potentil use in the preprtion of hemoglobin-bsed blood substitute or in preventing the precipittion of deoxygented hemoglobin S. To explore these possibilities, hemoglobin hs been treted with the polynion citrte in the presence of crbodiimide nd effects on structure nd some functionl properties hve been exmined. MATERIALS AND METHODS Hemoglobin nd hemoglobin S were prepred from humn blood obtined from the Cndin Red Cross. The Millipore Pellicon or Minitn systems were used to prepre concentrted sterile hemoglobin solutions, in Krebs-Ringer bicrbonte, whichwere stored t -80 C (Lng et l., 1990). Dilysis ginst 0.1 M MOPS djusted to the required ph with sodium hydroxide ws crried out prior to modifiction studies. Hemoglobin concentrtions were determined using Corning Co-Oximeter nd djusted to required concentrtions with MOPS buffer prior to tretment with citrte. Rections were initited by the ddition of the wter-soluble crbodiimide l-ethyl-3- (3-dimethylminopropyl)crbodiimide (EDC) obtined from Pierce sites of chemicl modifiction. To exmine the effect of the modifiction on the intct protein, t time intervls smples were diluted 5-fold into ice-cold 0.5 M Tris-HC1 buffer, ph 8.5, nd dilyzed ginst 0.1 M Tris-HC1 buffer, ph 8.5. Ion exchnge chromtogrphy ws crried out on the dilyzed mteril using Merck Superperformnce 150 mm X 10-mm DEAE column t flow rte of 1 ml/min. Rdioctivity ws determined in eluted frctions by scintilltion counting of liquots collected in frction collector t time intervls. Absorbnce of the eluted mteril ws monitored t 280 nm. For kinetic studies nd for peptide mpping, rections were stopped t time intervls by dilution of rection mixtures into ice-cold trichlorocetic cid. Pellets of precipitted protein were obtined by centrifugtion for 5 min in n Eppendorf centrifuge. The pellets were wshed with cetone nd redissolved in 70% formic cid. Rdioctivity in the formic cid-soluble mteril ws determined by scintilltion counting of liquots. Prior to peptide genertion formic cid-soluble mteril ws diluted into wter nd lyophilized. Peptides were generted in 0.1 M NH4HC03 by digestion with trypsin for 6 h t 37 C nd subsequently seprted by thin lyer electrophoresis on Merck cellulose pltes. Rdioctive peptides locted by rdioutogrphy were identified fter further purifiction using reversed phse HPLC on Zorbx ODS (Du Pont) nd FPLC on Superdex 75 (Phrmci) columns. Amino cid compositions were determined fter hydrolysis

2 nd mino cid nlysis on Beckmn System 6300 High Performnce Anlyzer. Exmintion of the effect of modifiction of hemoglobin with citrte on the content of primry mino groups in hemoglobin ws crried out using fluorescence mesurement (Peterson, 1983) fter rection with o-phthlldehyde (OPA). Globin ws prepred from smples treted with citrte nd from control smples in which either citrte or EDC hd been omitted from the rection mixtures. Weighed mounts of globin were dissolved in wter. Aliquots were llowed to rect with OPA nd the fluorescence in 0.5 N NOH ws subsequently determined s described for OPA determintion of intct proteins (Peterson, 1983). To determine totl protein contents, liquots were mixed with n equl volume of 12 N HCl nd seled in glss tubes. Hydrolysis ws crried out for 16 h t 106 "C. The cid ws then removed using SpeedVc concentrtor (Svnt Instruments). The dried hydrolyzed smples were dissolved in wter nd fluorescence fter rection with OPA ws determined s before nd compred with the fluorescence per gg of hydrolyzed bovine serum lbumin. Kinetic exmintion of rtes of incorportion of rdioctivity used procedures pproprite to studies of the modifiction of proteins in which sets of functionl groups with different rectivities could be present (Freedmn nd Rdd, 1968; Anderson nd Perhm, 1970). Oxygen binding properties of hemoglobins were determined using Hem-0-Scn (Americn Instrument Co.). Smples were dilyzed ginst Krebs-Ringer bicrbonte nd oxygen binding ws determined in the presence of 5.6% crbon dioxide. The pentcyclohexylmmonium slt of BPG (Sigm) in the sme buffer t finl concentrtion of 10 mm ws used to determine effects on the oxygen binding properties of modified nd control hemoglobin. Hemoglobin S solubility ws determined in solutions of 2.3 M potssium phosphte, ph 7.0, t 32 "C in the presence of 10 mg/ml sodium dithionite. Concentrted protein solutions were diluted into the buffer t room temperture. Turpidity leding to n increse in light scttering ws mesured t 650 nm in 1-cm light pth wter jcketed cell in Hitchi 124 Spectrophotometer. RESULTS The Effect of Citrte on the Oxygen Binding Properties of Hemoglobin-Preliminry experiments indicted ththe presence of citrte decresed the oxygen ffinity of hemoglobin. When present t concentrtion of 25 mm in 1 mm hemoglobin solution, citrte incresed the p50 vlue from 17 to 30 mm Hg s determined in Krebs-Ringer bicrbonte using the Hem-0-Scn. There ws no effect on co-opertivity of binding s the Hill coefficient ws 2.8 in both cses. This is similr to previous reports of reduced oxygen ffinity in the presence of liphtic poly(crboxy1ic cids) (Shimizu nd Bucci, 1974). However, when hemoglobin ws treted with citrte in the presence of the crboxyl-ctivting crbodiimide, the effect on oxygen binding ws much different. Fig. 1 shows the oxygen binding curves obtined when 1 mm hemoglobin ws incubted with 32 mm citrte for 1 h t 0 "C in 0.1 M MOPS buffer, ph 7.2, with nd without 26 mm EDC nd then dilyzed overnight ginst lrge excess of Krebs- Ringer bicrbonte prior to the determintion of oxygen binding. The curve for hemoglobin incubted in the bsence of EDC indictes tht citrte ws removed during dilysis s the mteril hd p50 of 17 mm Hg. Hemoglobin treted with citrte in the presence of EDC clerly showed ltered oxygen binding properties. Although the p50 vlue ws similr to tht of untreted hemoglobin, the binding curve ws much less sigmoidl. The Hill coefficient decreses from 2.8 to 1.3. The llosteric effector BPG hd no effect on citrte-modified hemoglobin oxygen binding. An identicl oxygen binding curve ws obtined for the modified mteril described in Fig. 1 when 10 mm effector ws present. This concentrtion of effector cused right shift of the oxygen binding curve of the control hemoglobin to p50 of 25 mm Hg. When hemo- globin ws treted with EDC in the bsence of citrte s described, no effect on the oxygen binding ws subsequently observed. The effect on oxygen binding seems therefore to be Modifiction Citrte of Hemoglobin PO, (mmhg) FIG. 1. Oxygen ffinity of hemoglobin nd modified hemoglobin. Hemoglobin (1 mm) in 0.1 M MOPS buffer, ph 7.2,ws incubted for 1 h with 32 mm citrte in the bsence (-) nd presence (- - -) of 26 mm EDC. After dilysis ginst Krebs-Ringer bicrbonte buffer, oxygen binding of hemoglobin solutions s function of oxygen concentrtion ws determined t 37 "C in gs mixtures contining 5.6% C02 using Hem-0-Scn Oxygen Dissocition Anlyzer. consequence of covlent ttchment of citrte to hemoglobin due to ctivtion of the crboxyl groups of the citrte by the crbodiimide nd subsequent covlent bonding to functionl groups on the protein. Effect of Citrte Modifiction on the Chromtogrphic Properties of Hemoglobin-When untreted purified hemoglobin tht hd been dilyzed ginst 0.1 M Tris-Cl buffer, ph 8.5, ws pplied to Merck Superformnce DEAE column equilibrted with 0.5 M Tris-C1, ph 8.5, single symmetricl bsorbnce pek ws observed in the elute fter 7.5 min when the column ws eluted t flow rte of 1 ml/min with 0.5 M Tris, ph 8.5. Pretretment of hemoglobin (0.5 mm) with EDC lone (26 mm) or citrte lone (32 mm) for 24 h t 0 "c in 0.1 M MOPS, ph 7.2, followed by dilysis ginst 0.1 M Tris-C1, ph 8.5, did not hve ny effect on the elution profile from DEAE under these chromtogrphic conditions. However, hemoglobin treted with both citrte nd EDC for 24 h under these conditions nd subsequently dilyzed ginst 0.1 M Tris-C1, ph 8.5, ws more strongly bound to the DEAE column, It could be eluted by mking the ph 8.5, 0.5 M Tris- C1 buffer 0.5 M in NC1. Fig. 2 describes elution conditions tht were developed to seprte unmodified nd modified hemoglobins on the DEAE column. The DEAE elution profile of hemoglobin tht hd been modified by citrte rdiolbeled with 14C in the presence of EDC for 10 min prior to dilution nd dilysis is shown. The first bsorbnce pek corresponded in elution time to unmodified hemoglobin. It contined 21% of the detected bsorbnce. An bsorbnce pek t 9.6 min with triling shoulder ccounted for 72.1% of the detected bsorbnce. Smller peks were detected t elution times of 24.5 nd 26.8 min, ccounting for 5.6 nd 8.9% of the detected bsorbnce, respectively. The recovered rdioctivity ccounted for 97% of the rdioctivity pplied. Two rdioctive peks were detected corresponding to the bsorbnce peks t 9.6 min (66% of the pplied rdioctivity) nd t 26.8 min (30% of the pplied rdioctivity). These findings re consistent with rpid limited incorportion of citrte into hemoglobin producing form of the protein slightly more negtively chrged thn the untreted protein under the chromtogrphy conditions nd therefore hving modertely incresed elution time. An ccompnying lower rte, but more extensive modifiction, could ccount for the strongly bound

3 Citrte Modifictionof Hemoglobin W n m 0.1 N ELUTION TIME (MINUTES1 I P FIG.2. Elution profile from DEAE of hemoglobin modified with citrte. Hemoglobin (0.5 mm) in 0.1 M MOPS buffer, ph 7.2, ws incubted in the presence of 32 mm citrte nd 26 mm EDC for 10 min t 0 "C. After dilution into 0.5 M Tris-CI buffer, ph 8.5, nd dilysis ginst 0.1 M Tris-CI buffer, ph 8.5, the smple ws pplied to Merck Superformnce DEAEcolumn. Grdient elution t flow rte of 1 ml/min ws used. Buffer A consisted of 0.5 M Tris-CI, ph 8.5, nd Buffer B consisted of 0.5 M Tris-CI, ph 8.5, contining 0.5 M NCI. The elution conditions consisted of 100% Buffer A for 10 min, liner grdient to 100% B over 10 min, 2 min of 100% B, liner grdient to100% A over 2min, nd subsequent elution t 100% A. Absorbnce t 280 nm detected using Bio-Rd model 1305 UV Monitor ws recorded using Beckmn model 427 Integrtor. Rdioctivity in the elute ws determined by scintilltion counting of liquots of 1-min frctions. rdioctive mteril elutedt 27.0 min. The smll bsorbnce pek t 24.5 min tht did contin rdioctivity ws not identified but could be free heme. Additionl support for rpid initil limited incorportion of citrte intohemoglobin ccompnied by slower rte, more extensive, incorportion ws obtined from DEAE chromtogrphy of mteril tht ws modified by citrte in the presence of EDC for longer times. One hour of modifiction under the sme conditions s for the 10-min modifiction produced n elution profile in which 91% of the detected bsorbnce nd 96% of the recovered rdioctivity ws found in pek elutingin 27.0 min. The remining 9% of the bsorbnce nd 4% of the rdioctivity ws found between elutiontimes of 7 nd 14 min. Rdioctivity detectedccounted for 98% of tht pplied. Chromtogrphy of mteril which hd been modified for 24 h under the sme conditions produced single mjor pek t 27.0 min, ccounting for 98.5 of the detected bsorbnce pplied nd 99% of the pplied rdioctivity. Determintion of the Sites of Modifiction-The nture nd extent of citrte modifiction of hemoglobin in the presence of EDC ws further exmined fter digestion of modified protein with trypsin ndsubsequent seprtions of peptides. Fig. 3 shows rdioutogrph of tryptic peptides generted from hemoglobin tht hd been incubted with rdioctive citrte. It cn be seen tht two rdioctive bnds pper fter short period of rection. Other bnds indictive of slower recting mteril pperfter more extensive incubtion, but the rpidly lbeled bnds were still present. Sincethe rpidly lbeled bnds were present fter extensive times of modifiction, the subsequent formtion of other lbeled mteril ORIGIN I) I. 2. I,:-, lomlnutes 24HOURS 7p.72H p. 2H FIG.3. Rdioutogrph of tryptic peptides. Hemoglobin (0.5 mm) ws incubted with 32 mm ["Clcitrte, specific rdioctivity 0.3 mci/mm, in 0.1 M MOPS buffer, ph 7.2, t 0 "C in the presence of 26 mm EDC for the times indicted.wshedtrichlorocetic cid pellets were digested for 6 h t 37 "C with trypsin (20 pg/mg hemoglobin) in 0.1 M NH,HCO3. After drying undervcuum, smples were dissolved in cetic cid:formic cid:wter, 82:90, ph 2.1, nd electrophoresis ws crried out for 1 h t 500 V on cellulose pltes wetted with the sme solvent usingc-dinitrophenyl lysine spotted t the origin s colored mrker. After electrophoresis, exposure of the pltes to Kodk X-Omt AR film for5dys ws used to locte rdioctivity. does not pper to be due to further rection of the initilly lbeled groups to form cross-links. Acrylmide gel electrophoresis in sodium dodecyl sulfte of mteril tht hd been extensively modified nd shown to contin multiple bnds when tryptic digests were electrophoresed nd rdioutogrphed gve no indiction of subunit cross-linking. Further purifiction of the rpidly lbeled lower mobility rdioctive bnd (m = 0.88 versus mrker) on Superdex 75 FPLC column followed by reverse phse HPLC on Zorbx peptide column gve mteril which hd n mino cid composition of Asp (0.9), Ser (0.9), Al (0.8), Vl (l.o), Leu (1.01, Pro (1.0) when normlized for Lys content. No other mino cids were present in significnt mounts. The composition is identicl to tht of tryptic peptide rising from the Nterminl region of -globin (Vl-Leu-Ser-Pro-Al-Asp-Lys), nd it ws therefore concluded tht one of the residues rpidly lbeled with citrte in the presence of EDC ws the Nterminl vline of 0-globin chins. From the higher mobility rpidly lbeled rdioctive bnd (m = 1.22 versus mrker) similrly purified mteril ws found to hve composition of Thr (0.9), Glu (1.7), Vl (LO), Leu (l.o), His (1.01, Pro (LO), with no other mino cids presentin significnt mounts when normlized for Lys content. This composition is identicl to tht of tryptic peptide rising from the Nterminl region It ws therefore concluded tht thesecond residue tht rected rpidly with citrte in the presence of EDC ws the N-terminl vline of 8-globin chins. Other incorportion of rdioctivity is presumbly due to slower rection of mino

4 groups from lysine residues with citrte in the presence of EDC. Effect of Citrte Modifiction of Hemoglobin on Subsequent Rection o-phthlldehyde-chromtogrphy on DEAE nd peptide mpping indicted tht slow rte modifiction of number of functionl groups in ddition to the N-terminl mino groups occurred when hemoglobin ws llowed to rect with citrte in the presence of EDC for long periods of time. The extent of this modifiction nd n indiction of the nture of the recting groups were exmined in greter detil using regent tht rects with primry mino groups with the subsequent production of fluorescence species which cn be quntified. The fluorescence produced when globin prepred from hemoglobin tht hd been modified with citrte for 24 h under the conditions described in Fig. 3 ws rected with OPA ws compred to the fluorescence produced when identicl mounts of globins from hemoglobin treted with citrte in the bsence of EDC or EDC in the bsence of citrte were rected with OPA. Globin from the modified hemoglobin produced 49.3 k 2.0 fluorescence units/pg protein. Globin from hemoglobins in which only EDC or citrte were present during the rection produced 68.0 f 2.6 fluorescence unitslpg protein. These findings re comptible with the rection of 27.5% of the primry mino groups of hemoglobin with citrte in 24 h under the rection conditions described in Fig. 3. Kinetics of Citrte Incorportion into Hemoglobin-Preliminry experiments were crried out to determine conditions tht would llow the kinetic exmintion of the incorportion of rdioctivity from citrte into hemoglobin. The most common ppliction of crbodiimides in protein modifiction studies is to ctivte the protein crboxyls tht subsequently rect with dded nucleophilic regents or with nucleophilic functionl groups within the protein. In the present cse, crbodiimide ctivtion of citrte crboxyls occurs under conditions in which hemoglobin crboxyl groups would lso be expected to be ctivted. It ws therefore decided to use limiting mounts of the crbodiimide with view to selecting for ctivted crboxyl groups most rective with nucleophilic functionl groups in hemoglobin. Initil experiments were crried out t ph 7.2. At constnt crbodiimide concentr- tions, citrte nd hemoglobin concentrtions were vried until conditions were found under which the mount of rdioctivity incorported fter extensive incubtion depended directly on the mount of hemoglobin present. At n EDC concentrtion of 26 mm nd citrte concentrtion of 32 mm, citrte incorportion into hemoglobin fter 24-h incubtion t 0 "C incresed in liner fshion with hemoglobin concentrtions from 0.25 to 1.0 mm. The time course of the incorportion of citrte into hemoglobin ws then determined under these conditions nd nlyzed ssuming first order kinetics. Liner semi-logrithmic plots were tken s evidence tht the nlysis method ws pproprite. Fig. 4 shows the plot of dt obtined in n experiment in which trichlorocetic cid-insoluble rdioctivity ws determined t intervls with the rdioctivity incorported t 20 h tken s the mximum incorportion. The best fit line obtined by liner regression hs n R vlue of The first order rte constnt given by the slope is min". However, it cn be seen tht the line does not pss through the origin. This could be explined by the presence of some rective groups, which rect completely very quickly compred to other groups. From the specific rdioctivity of the citrte used, the mount of citrte incorported t the much fster rte cn be clculted. From this nd the mount of hemoglobin present, it ws clculted tht 3.85 mol of citrte/mol of hemoglobin or 0.96 mol of citrte/ mol of globin chin ws rpidly incorported. Together with Citrte Modifiction of Hemoglobin I TIME (minutes) FIG. 4. Rte of Incorportion of rdioctivity from ["C] citrte into hemoglobin. Hemoglobin (0.3 mm) ws incubted with 32 mm citrte (0.05 mci/mmol) in the presence of 26 mm EDC in 0.1 M MOPS buffer, ph 7.2, t 0 "C. Aliquots (20 pl) were tken in triplicte t time intervls, nd rdioctivity ws determined by scintilltion counting. The incorportion of rdioctivity ws ssumed to be complete fter 20 h. The nturl log of the rtio of rdioctivity incorported fter 20 h (Rt) to the rdioctivity incorported fter 20 h minus the rdioctivity incorported t specific time intervls (R,- R,) is plotted uersus the time intervls t which liquots were tken. The slope indictes the first order rte constnt, nd the intercept on the y xis indictes the portion of rdioctivity incorported t much fster rte thn tht given by this rte constnt. the other dt on the sites nd extent of modifiction, this suggests tht N termini of both - rect with citrte very rpidly compred to set of other groups, probbly mino groups contributed by lysine residues, ll of which pper to rect t similr rtes to ech other. Further exmintion of the rectivity of functionl groups in hemoglobin with citrte ws crried out by determining the rdioctivity in specific tryptic peptides generted from hemoglobin liquots tken t intervls throughout incubtion. After seprtion of peptides by thin lyer electrophoresis, the rdioctivity in regions from which peptides could be isolted corresponding in composition to the N-terminl tryptic peptides of - ws determined. There ws no difference detectble in the mount of rdioctivity in the two regions over the,time intervls nd the incorportion of rdioctivity ws colhplete fter 20-min incubtion. Fig. 5 shows the plot obtined using the rdioctivity incorported fter 20 min s the: mximum. The R vlue for the line obtined by liner regression is 0.99 nd comes very close to pssing through the origin. The first order rte constnt given by the slope of the line is min". The rte of rection ws incresed by incresing the hydro- gen ion concentrtion. Under conditions identicl to those used to generte the dt of Figs. 4 nd 5 except tht 0.1 M MOPS t ph 6.6 ws used insted of 0.1 M MOPS, ph 7.2, nd nlysis of dt s described, the rte constnts clculted from slopes were min" for the slow group nd min-' for the fst group of N-terminl residues. When the rection ws crried out s before but in 0.1 M MOPS, ph 7.8, the rte of incorportion of rdioctivity decresed. Rdioutogrphs of tryptic digests did not indicte more rpid incorportion into the N-terminl peptides. The kinetic nlysis indicted tht first order single rte constnt of min" pplied to ll the rective groups. Modifiction of Hemoglobin S-Incorportion of citrte r- dioctivity into hemoglobin S ws the sme s incorportion into norml hemoglobin when crried out under identicl

5 15508 Modifiction Citrte of Hemoglobin TIME (rnmutes) FIG. 5. Rte of incorportion of rdioctivity into the rpidly recting functionl groups of hemoglobin. Incubtion conditions with ["Clcitrte were the sme s described in Fig. 4, nd the dt were plotted in the sme wy. Aliquots were tken in triplicte t time intervls. Tryptic peptides were generted from trichlorocetic cid-insoluble mteril obtined from ech liquot nd subsequently seprted nd detected s described in Fig. 3. Rdioctivity in the rpidly lbeled bnds ws quntitted by scintilltion counting fter elution from scrped res. The rdioctivity in the res from which the N-terminl peptides cn be purified reched mximum in liquots tken fter 20 min incubtion. This mount of incorported rdioctivity ws used s R, C 0 In '0 0.4 w 0 z m 0.3 m / [HbS,.,....., , rngirnl HbS 0.42 rnpiml HbS 0 42 rnglml HbS cit C.-.-.-"' TIME (MINUTES) FIG. 6. Time course of the development of turpidity in hemoglobin S solutions. Hemoglobin S t concentrtions of 0.42 mg/ ml (.-.) or 0.82 mg/ml (- - -) or mixed with citrte-modified hemoglobin S (0.42 mg/ml mg/ml) (-) ws incubted in 2.3 M potssium phosphte buffer, ph 7.0, t 32 "C. The increse in bsorbnce due to light scttering ws monitored for 1-cm light pth t 650 nm. conditions. The effect of modifiction with citrte on the solubility of deoxygented hemoglobin S is shown in Fig. 6. Dilute solutions of hemoglobin S ggregted rpidly when incubted in the deoxy form t 32 "C in concentrted phosphte. Incresing the protein concentrtion decresed the time required for the onset of ggregtion. However, if citrtemodified hemoglobin S ws present, the ggregtion of unmodified hemoglobin S ws impeded. Solutions of only citrtemodified hemoglobin S showed no evidence of ggregtion t concentrtions of up to 1 mg/ml for t lest 1 h t 32 "C in concentrted phosphte. DISCUSSION The use of crbodiimide ctivtion in protein modifiction studies hs minly involved pplictions in which n exogenous nucleophilic mine is dded in lrge excess to proteins in which crboxyl residues re ctivted resulting in the covlent ttchment of the mine to the protein (Crrwy nd Koshlnd, 1972). At high concentrtions, EDC hs been used to rect with protein functionl groups in the bsence of dded exogenous nucleophile (Verburg et l., 1992). High concentrtions of EDC my lso result in protein cross-links (Mejillno nd Himes, 1991). In this study different pproch hs been used. The crboxyl groups of n exogenous regent re ctivted with crbodiimide nd llowed to rect with nucleophilic protein functionl groups t close to neutrl ph. It hs been shown tht citrte cn be used to modify hemoglobin with high degree of selectivity with respect to the functionl groups of hemoglobin with which it rects. Citrte is highly negtively chrged t the ph vlues t which selective modifiction hs been shown to occur nd cn decrese the oxygen ffinity of hemoglobin presumbly by binding to the positively chrged region However, crbodiimide-ctivted citrte t less thn physiologicl ph rects preferentilly with the CY mino groups of the N- terminl residues of - There re number of possible explntions for this. Activtion of crboxyl by cr- bodiimides leds to positively chrged dduct (Crrwy nd Koshlnd, 1972). Ionic interctions leding to citrte binding to the protein my not be the most importnt fctor. The CY mino groups my be more nucleophilic or more ccessible to the regents under the conditions used. Exmintion of citrte rection with other proteins under conditions similr to those tht hve been used for hemoglobin my be useful in determining the mjor fctors involved in the rection. In the present study, the incresed of rte rection observed with incresing hydrogen ion concentrtion is to be expected since formtion of the crbodiimide-crboxyl dduct requires protontion (Crrwy nd Koshlnd, 1972) nd the crbodiimide would be expected to be reltively stble over the ph rnge used (Gilles et l., 1990). Conditions hve been developed under which limited modifiction of hemoglobin with citrte cn be obtined. At ph vlues ner neutrlity with defined concentrtions of rectnts, limited incorportion of citrte into two sets of functionl groups bsed on rtes of rection cn be obtined. The conclusion tht the functionl groups recting rpidly with citrte in the presence of EDC re the N-terminl mino groups of the CY- is bsed on direct evidence. When [14C]citrte ws used, there ws rpid rdiolbel incorportion into mteril produced by tryptic digestion, which, when purified nd hydrolyzed, corresponded in mino cid composition to the mino cid compositions of the N-terminl peptides tht would be expected from tryptic digestion of Rtes were determined directly on this mteril fter electrophoretic seprtion of peptides. It seems likely tht the more slowly recting groups obtined under the rection conditions used represent set of lysine residue mino groups. The chromtogrphic properties on DEAE of hemoglobin fter extensive times of modifiction suggest highly negtively chrged molecules s would be produced by replcement of lysine mino group positive chrges by negtive chrges from crboxyl groups of coupled polyn- ionic citrte. Peptide mps nd OPA tretment of mteril produced under the rection conditions specified indicte tht

6 Hemoglobin of Modifiction Citrte only limited number of groups rected. The tryptic peptide centrted solutions of hemoglobin S (Adchi et l., 1987). mps contined fewer bnds thn would be expected if ll 11 Accordingly, retrdtion of ggregtion of dilute solutions lysine mino groups of -globin nd ll 11 lysine mino groups suggests conditions tht might led to the retrdtion of of 0-globin rected equivlently. After 24 h of modifiction t geltion of concentrted solutions in red blood cells. The ph 7.2 with citrte under the define concentrtion conditions, ltered conditions used in the present study to detect ggre- 72.5% of the OPA-rective mteril remined. gtion in which room temperture (22 "C) solutions were When modified to limited extent with citrte described, s wrmed to 32 "C nd nitrogen flushing ws eliminted give hemoglobin co-opertivity with respect to oxygen binding is similr responses in tht the onset of turpidity occurs more lost. Although the p50 of citrte-modified hemoglobin is sim- rpidly with incresing temperture nd incresing hemogloilr to tht of unmodified hemoglobin, the oxygen ffinity t bin S concentrtion. Since ggregtion of deoxy hemoglobin oxygen tensions relevnt to physiologicl situtions is lower. S ws clerly retrded in mixtures of hemoglobin S nd Similr effects on oxygen binding hve been reported fter hemoglobin S modified with citrte nd the modified protein crboxymethyltion of N-terminl residues of hemoglobin by itself showed no indiction of ggregtion under conditions (Fntl et l., 1987). Limited citrte modifiction bolishes the in which hemoglobin S showed substntil ggregtion, citrte response of hemoglobin to BPG. This cn be ccounted for modifiction my prove to be useful method to prevent geltion. by the proximity of the LY mino group of P-globin to the binding site for this regultor in the cvity between P chins (Perutz et l., 1980). It would be of interest to determine the effects of citrte modifiction on crbon dioxide binding (Vndegriff et l., 1991). Citrte contins three crboxyl groups. It is therefore pos- sible tht fter rection with the N-terminl mino group further rection leding to intr- or intersubunit cross-linking could occur involving n dditionl crboxyl group of the citrte. However, there ws no evidence of cross-linking of either type when rections were crried out s described. If the modified hemoglobin were to be developed s potentil oxygen-delivering blood substitute n dditionl step which in subunits would be covlently linked would be necessry. The free crboxyl groups incorported from citrte could possibly be exploited for this purpose. The preliminry dt on the properties of hemoglobin S modified with citrte suggest tht the modifiction could hve vlue in preventing the ggregtion to form gel tht cuses sickling when it occurs inside red blood cell. The method used to determine hemoglobin S solubility is similr to procedure in which deoxygention is crried out in n nerobic cell flushed by nitrogen nd the temperture is rised from 0 to 30 "C nd the time to onset of incresing turpidity is mesured t 700 nm (Adchi nd Askur, 1979). Aggregtion of dilute solutions of hemoglobin in concentrted phosphte solutions under these conditions hs been proposed to be similr to the mechnism leding to the geltion of con- Acknowledgment-Amino cid nlysis ws crried out t the Children's Hospitl of Estern Ontrio with the kind permission of Dr. Hns Heick. REFERENCES Adchi, K., nd Askur, T. (1979) J. Biol. Chem. 254, Adchi, K., Kim, J., Kinney, T. R., nd Askur, T. (1987) J. Biol. Chem. 262, Anderson, P. J., nd Perhm, R. N. (1970) Biochem. J. 117, Benesch, R. E., nd Kwong, S. (1988) Biochem. Biophys. Res. Commun. 156, 4-1 " " A Berbers, G. A. M., Bleeker, W. K:, Stekkinger, P., Aglerberg, J., Rigter, G., nd Bkker, J. C. (1991) J. Lb. Ch. Med. 117, Crrwy, K. 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