Covalent fixation of polymer-linked benzene hexacarboxylate onto human haemoglobin Mich~le L~onard and Edith Dellacherie* Laboratory of Macromolecular Physical Chemistry, URA CNRS 494, ENSIC, BP 451, 54001 Nancy Cedex, France

(Received 3 January 1991; revised 11 April 1991) The reaction of human deoxy and oxyhaemoglobin with a macromolecular effector, monomethoxypolyoxyethylenelinked benzene hexacarboxylate, in the presence of a water soluble carbodiimide, produces under defined conditions, the same conjugates preferentially acylated at the two valines ill. The oxygen affinity of both these conjugates is decreased by approximately 5-fold compared with that of native Hb (at pH 7.2, in 0.05 M Tris buffer, 25°C, Ps0:20.1 and 20.7 Torr versus about 4 Torr for Hb). This difference appears to be due to an overstabilization of the T state probably together with a decrease of the oxygen affinity of the R state. Addition of IHP to the conjugate solutions does not influence the Pso but addition of IHP to the reaction mixtures before the couplino limits the substitution of lib by the macromolecular effector, to 20% (instead of 100% in absence of IHP ). The cooperativity curve is shifted to the rioht with an nmax of 3 at about 90% oxygen saturation, which corresponds to a potential release of 48% of oxygen at pH 7.2, 25°C, between 100 and 40 Torr, compared with 40% for blood. Such kinds of conjugates especially those obtained from oxyhaemoglobin which are easily prepared, could be of a great interest as non-diffusing oxygen carriers in transfusional and perfusional fluids. Keywords: Oxyhaemoglobin;oxygen affinity;covalent fixation

The reaction of deoxy H b t with pyridoxal phosphate, first described by Benesch et al. 1, leads, under certain conditions, to derivatives with a lowered oxygen affinity compared with free Hb and a nearly unchanged cooperativity. Due to this particular property which is of great interest in the field of artificial oxygen-carrying resuscitation fluids 2'3, a large number of papers have been devoted to pyridoxylated Hb 3'4 and to Hb modified with other anionic derivatives 5 -9 The low oxygen affinity of such covalent adducts can first be related to the fact that Hb is modified inside its polyphosphate binding site by compounds that possess negatively charged moieties. These moieties, by lowering the net positive charge of this fl cleft and by forming new salt bridges in deoxyHb with the N H 3 + of this site, could be responsible for an overstabilization of the deoxy conformation 3- 9. Another explanation for the decreased oxygen affinity in the case when c~l valines are substituted 5'6, is that the small anionic moiety creates, at this site, a high local density of negative charge and mimics the effect of chloride ions in lowering the Hb affinity for oxygen. Another class of low oxygen affinity derivatives of Hb can be obtained by chemically crosslinking either the polyphosphate binding site with negatively charged

* To whom correspondenceshould be addressed. f Abbreviations:Hb, human adult haemoglobin;BHC-MPOE, monomethoxypolyoxyethylene-linkedbenzene hexacarboxylate; Hb-BHCMPOE, human haemoglobincovalentlybound to monomethoxypolyoxyethylene-linkedbenzenehexacarboxylate;BHC,benzenehexacarboxylate; EDCI, N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimidehydrochloride;2, 3-DPG,2,3-diphosphoglycerate;IHP, inositolhexaphosphate; TPCK,L-l-tosylamido-2-phenylethylchloromethylketone trypsin. 0141-8130/91/050266-07 © 1991 Butterworth-HeinemannLimited 266 Int. J. Biol. Macromol., 1991, Vol. 13, October

difunctional reagents x°- 13, or another site of Hb located between the two a-chains (Lys ~1 99 and Lys ~2 99) by means of bis(3,5-dibromosalicyl) fumarate ~4. However, some of these derivatives used in infusions on rats as acellular blood substitutes exhibited retention times only 2 to 4 fold greater than that of Hb s'l 5 probably because the tetramer, even when stabilized by intramolecular crosslinking, is still capable of filtering through the somatic capillary beds. Our attempts to synthesize polymeric Hb conjugates with low oxygen affinity and high molecular weight 16, aimed to prepare macromolecular effectors of Hb and to bind them to the protein. In the course of our studies, we observed that polyanionic derivatives of dextran, such as phosphates and sulphates, specifically interacted with deoxyHb ~7'18 and that their covalent coupling to it led to low oxygen affinity conjugates 17. Other dextran derivatives bearing benzene polycarboxylate units were also found to lead to low oxygen affinity conjugates when linked to deoxyHb 17 but quite surprisingly, similar results were obtained when they were linked to oxyHb 19. Generally, according to the literature, the chemical modification of deoxyHb is necessary in order to produce low oxygen affinity derivatives 3'4'9- 14 except when ~1 valines are involved 5- 7 or in the case of the fixation of mono-(3,5-dibromosalicyl) fumarate s. To explain the results obtained by coupling our dextran-linked benzene polycarboxylates to oxyHb, we prepared another polymeric derivative from polyoxyethylene with a structure schematically described in Figure 1 and bearing only one linked benzene hexacarboxylate per chain at its extremity ( B H C - M P O E ) unlike the dextran derivative that was randomly polysubstituted and investigated its ionic and covalent

Covalent fixation of benzene hexacarboxylate onto human haemoglobin: M. Ldonard and E. Dellacherie

HOCO COOH CH3-(OCH 2 C H 2 ) n - N H - C O - ~ ) - C O O H BHCoMPOE

+

H 2 N-Hb

HOCO COOH

(COOH)4 EDCI . CH 3 .(OCH2 Ctt2 )n - N H - C O - ~ ) CO-NH-Hb

Figure 1 Schematic representation of the reaction of Hb with monomethoxypolyoxyethylene-linkedbenzene hexacarboxylate association with Hb (Figure 1). Here, we report on the structural and functional characterization of the HbBHC-MPOE covalent conjugates.

Experimental Materials and chemicals Human Hb was prepared from outdated blood as previously described 2°. BHC-MPOE (average molecular weight 5000) was synthesized and purified according to the recently published procedure 21. EDCI was purchased from Aldrich (Belgium), IHP (sodium salt) and trypsin (TPCK treated) from Sigma (USA), ultra-pure urea from Merck (FRG). Carboxymethylcellulose (CM 52) was obtained from Whatman (England). The gel filtration chromatographic experiments were performed either under low pressure with AcA 54 and AcA 202 Ultrogels (from IBF, France) or high pressure with a TSK G3000 PW column, 60 cm (from LKB, Sweden). High performance reverse phase chromatography was carried out on a Brownlee column, Aquapore RP 300, 25cm (from Touzart et Matignon, France). Covalent fixation of BHC-MPOE onto Hb Fifteen ml 10% Hb solution (23/~mol) were added to 20 ml of water and the pH was adjusted to 5.8 by adding 0.1 M HCI. Then 300 mg of BHC-MPOE (with 156 pmol linked BHC/g polymer) and 8.8 mg of EDCI were added to the solution. The reaction mixture was stirred for 1 h at 5°C. The extent of modification of Hb was assessed by chromatography on AcA 54 (exclusion limit for proteins: 90 000) with 0.05 MTris buffer, pH 7.2, as eluent. The amount of unreacted BHC-MPOE was determined by h.p.l.c, on a TSK G3000 PW column (exclusion limit: 20 000 for polyethylene glycols and 100 000 for proteins), with 0.075 M Tris buffer, pH 7.2, as eluent. The reaction with deoxyHb was carried out under a nitrogen atmosphere in a glove box. The absence of oxygen in the Hb solution was checked spectrophotometrically. Oxygen-binding studies Before studying the oxygenation equilibria, the covalent conjugates were gel filtered on AcA 54 Ultrogel (eluent: 0.05M Tris buffer, pH 7.2). Routine oxygen equilibrium measurements were performed by the spectrophotometric method of Labie and Byckova22, using a tonometer. Measurements were made at 560 and 578 nm, in 0.05 M Tris buffer, pH 7.2, at 25°C. The concentration in Hb tetramer was 15/~M.The oxygen

pressure corresponding to the half-saturation of Hb (Pso) and the corresponding Hill number (ns0) were calculated by means of a simple program of least-square analysis. The Bohr effect was determined from the curve log Pso vs pH in 0.05 M Tris buffer, 0.1 ra NaC1, at 25°C. In all cases the percentage of methaemoglobin determined by the method of Kaplan 23 was found to be less than 5 after coupling. Extended oxygen binding curves were determined by an automatic continuous method (Hemox Analyzer, TCS, Southampton, PA, USA) in 0.05 M Tris buffer, pH 7.2, 25°C ([Hb] = 30 #M). The recording system of the oxygen-binding experiments was interfaced with an Apple microcomputer, programmed to store on tape up to 700 values of absorbance and Po2. The Pso and nso values were computed from the experimental points in the 40-60% saturation range by linear regression analysis. The experimental oxygen-binding curves were fitted to the Monod-Wyman-Changeux equation for y24 ( y = fraction of oxygenated Hb), using an iterative non-linear least-square program. Initial estimates of K R and K r, the oxygen dissociation constants for the R and T states respectively, were obtained from the Hill graph, assuming a slope of unity below 1 and above 99% oxygen saturation. L( = (Pso/KR) 4) is the equilibrium allosteric constant and c the KR/KT ratio. The curve giving the variation of Hill coefficient n versus log Y/1 - Y was computed at the first derivative of the Hill plots, according to a method described by Craescu et al. 25. Peptide mapping The conjugate samples, gel-filtered on AcA 54 Ultrogel and dialysed against 2 mM phosphate buffer pH 5.8, were applied to a column of CM 52 carboxymethylcellulose (1 x 20 cm) which was developed, at 5°C, with a linear gradient of sodium phosphate buffer pH 5.8 from 2 to 200 mM. The Hb derivatives were detected at 280 nm. These fractions were then separated into 0t- and r-chains following the method of Clegg et al. 26 at pH 6.9, on a CM 52 carboxymethylcellulose column (1 x 14 cm). In this chromatographic system, the fact that the benzene polycarboxylate groups were negatively charged made it possible to separate the modified globins from the unmodified ones. The globins were detected at 280 nm. As BHC-MPOE also absorbs at this wavelength, the percentages of modified ct and fl globins could not be calculated directly from the corresponding peaks; they were then calculated as the difference between the peak areas of remaining unmodified globins and the peak areas ofglobins produced by an amount of native Hb identical to that of the studied modified Hb. The separated ~- and r-chains were digested with trypsin (TPCK treated) and the tryptic peptides were analysed according to Wajcman 26a. Twenty mg of the modified globins (~ or r) were dissolved in 10 ml of 25 mu ammonium bicarbonate buffer, pH 8.8, and 200 #l of a trypsin-containing solution (1 g/l in 1 mM HCl) were added. The mixture was stirred for 2 h and another 200/d of the same trypsin solution were added. After 1 night at room temperature the mixture was freeze-dried. Five mg of the resulting soluble peptides were applied to a C s Aquapore RP 300 column, equilibrated with the following mixture: A/B, 90/10 vol; A: 0.05% aqueous trifluoroacetic acid; B: acetonitrile/0.1% aqueous trifluoroacetic acid, 50/50 vol. The column was developed

Int. J. Biol. Macromol., 1991, Vol. 13, October

267

Covalent fixation of benzene hexacarboxylate onto human haemoglobin: M. L~onard and E. Dellacherie within 60min with a gradient starting from the A/B mixture (90/10 vol) pure B, at a flow rate of 1 ml/min. The peptides were detected at 214 nm.

~O.D.

llb

280 nm

Amino acid composition

I

After tryptic digestion of modified globins, the polymer-linked peptides were separated from the unmodified ones and isolated by gel filtration chromatography on an AcA 54 Ultrogel column (1 x 30cm, 0.1 M NaC1, 30ml/h). After dialysis and freeze-drying, these high molecular weight samples were hydrolysed with 6 M HCI in vacuo for 15 h at 105°C, then dried and analysed on a Beckman 7300 amino acid analyser.

Vo

/

;

I

~

\

_

MPOE-BHC

Results

v,/Vo

Influence of BHC-MPOE on the oxygen-binding of Hb Figure 2 shows the effect on Hb Pso of BHC added in increasing concentration to an Hb solution. BHC is known 27'2s to be a strong effector of the protein as it provokes an increase of Hb Ps0 from 4 to 30 Torr for relatively low values of the molar ratio r = B H C / H b , i.e. 30 (for [ H b ] = 15/~M). Under the same conditions, the natural intraerythrocytic effector, 2,3-DPG led to a maximum Ps0 of 14 Torr. Compared to this effect, that of MPOE-linked BHC ( B H C - M P O E ) is less, as the Pso limit value obtained for r ~ 50 is 14 Torr (Figure 2), but it should be noted that linked BHC contains no more than five free carboxylate functions (whereas free BHC contains six). For example at pH 6, in 0.1 M bis-Tris/lactic acid buffer, 20°C, and for [ H b ] ~ 120#M, Desbois and Banerjee 27 report Pso values of 55 and 34 Torr for BHC and benzene pentacarboxylate respectively (molar ratio benzene polycarboxylate/Hb = 5; Pso for free Hb = 14 Torr).

Covalent fixation of BHC-MPO E onto oxy and deoxyHb Figure 3 shows the gel filtration elution profile on AcA 54 Ultrogel of the covalent conjugate prepared from oxyHb with 2mol B H C - M P O E / m o l Hb and 1 mol E D C I / m o l B H C - M P O E (conjugate A). The molecular weight distribution lies between two values corresponding respectively to the exclusion limit (Mr = 90000) and to

40 (Ton)

30

S

20

1

free Hb. The percentage of free Hb, although difficult to determine with accuracy, could be evaluated at less than 5. By eluting the conjugate in h.p.l.c, on a TSK G3000 PW column (detection at 254 nm), it was observed that no unreacted B H C - M P O E remained in the reaction mixture. The same profiles were obtained for the conjugate prepared from deoxyHb (conjugate B). When the coupling reaction was carried out in the presence o f l H P ( 10 mol I H P / m o l Hb), the elution profile of the reaction mixture was completely different, showing the largest fraction (more than 80%) at the elution volume of free Hb (Figure 3).

Oxygen-binding properties of conjugates The values of Pso, determined by the tonometer method, of the covalent conjugates H b - B H C - M P O E prepared from oxy- (A) and deoxyHb (B) are shown in Table 1. Both show a significantly decreased oxygen affinity relative to Hb, and the addition of I H P had no effect on the P5o values. The Hill plot of the oxygen-binding curve of conjugate A (determined by the automatic method) compared to that of Hb is presented in Figure 4a. A notable right shift, specially at the top of the Hill plot, is observed for modified Hb. The cooperativity curve (Figure 4b) shows that H b - B H C - M P O E still possesses a good cooperativity Oxygen-binding parameters of Hb-BHC-MPOE conjugates compared with those of Hb

I

Free Hb Ab Bb

Pso (Torr)

(Torr)

3.8 20.6 20.1

49.8 20.7 20.8

Pso a

1

Figure 2

Effect of free (11) and MPOE-linked (1--1)BHC on Pso of Hb; 0.05M Tris buffer, pH 7.2, 25°C, tonometric method. [Hb]: 15gM

268

1.6

Table 1

[BHC] (raM) o

1.4

Figure 3 Elution profile on AcA 54 Ultrogel of the covalent conjugate of BHC-MPOE with oxyHb ( - - - ) and of the reaction mixture obtained when IHP was added before the coupling reaction ( ). The arrows indicate the elution volumes of free Hb and unreacted BHC-MPOE

10

0

1.2

Int. J. Biol. Macromol., 1991, Vol. 13, October

Conditions: 0.05M Tris buffer,pH 7.2, 25°C; tonometric method a Values determined in presenceof IHP ( 10 mol/mol Hb) bA, B: conjugatesprepared from oxy- and deoxyHb respectively,with 2 mol BHC-MPOE/mol Hb and 1mol EDCI/mol BHC-MPOE

Covalent fixation of benzene hexacarboxylate onto human haemoglobin: M. L~onard and E. Dellacherie

2

Z.

..//,

.-. 1 Pse: ,9.~, ,sa: z.a61 ~_ 0

"6

M-; ', ', ' ~ '

set of data a single well-determined value of KT was obtained (43-44 Torr), but more than one value of KR and L generated curves with comparable standard errors. Anyway, KR was always found higher for conjugate than for Hb (between 0.46 and 0.64 Torr) as well as L (between 1.3 and 2.8 x 106).

"~

/!,,

~-i:l:I~4 : ',:',-4-~--~-:. . . . ~4-~-/-q--:-~ ~--bi-'-{-;~--~-~4 -

/

The determination of the Bohr effect Figure 5 illustrates the pH dependence of log Pso in both conjugates A and B, compared to that of free Hb. The conjugates exhibit a reduction of respectively 39.5 and 21% of the alkaline Bohr effect as AH ÷ max/haem increases from - 0 . 5 3 for Hb to - 0 . 3 2 and - 0 . 4 2 for A and B respectively.

/

2:/ ~2

log P0 z [Torr]

Cation-exchange chromatography of Hb conjugates The conjugates A and B, prepared from oxy- and deoxyHb respectively, were fractionated on CM 52. Figure 6a shows the profile obtained for A. For this conjugate, three species were eluted: A2, A 1 and A o, representing 15, 65 and 20% respectively. A o was identified as unmodified Hb. For conjugate B, the profile was the same with 3 peaks B2, B1 and B o, representing 20, 60 and 20% respectively (not shown). B o was unmodified Hb. A 2 and B 2 correspond to the most negative species, i.e. the most substituted. The presence of about 20% of unmodified Hb in the eluates is rather surprising, as it does not appear in the

R 3 :

/

.j

.v": '~''4"

/

b,

1,61 log P 50

-2

--1

0

1

2

tog(Y/I-Y)

1,4

Figure 4 (a) Hill plots of oxygen-binding curve for conjugate A (2 mol BHC-MPOE/mol Hb and 1 mol EDCI/mol BHCMPOE) compared with that of Hb (....). The curve has been fitted ( .... ) to a MWC equation as described in the text. (b) Hill coefficient n vs log Y/1 - Y for conjugate A. Conditions as in Table 2 with an nmax of about 3, but shifted to approximately 90% of oxygen saturation. The apparent values of the parameters of the two-state allosteric model and the oxygen dissociation constants in the T and R states are given in Table 2. The results indicate that for modified Hb, the dissociation constant Kx is higher than that of Hb, which is indicative of a stabilization of the T deoxystate; on the other hand, the Ks value of conjugate is only indicative since, as already mentioned x~,29, reasonably good fits of low square residuals were obtained with more than one set of parameters; for each

1,2 1,0 0,8 0,6 0,4

5

I

I

6

7

pH

8

Figure 5 Oxygen Bohr effect for conjugates A (A) and B (A) and for Hb ( • ) ; 0.05 MTris buffer, 0. l MNaC1, 25 °C, tonometric method

Table 2 Oxygen-binding and allosteric parameters of Hb-BHC-MPOE prepared from oxyHb

Hb Hb-BHC-MPOE

Ps0 a (Torr)

nso

KR (Torr)

KT (Torr)

L

c

4.2 20.7

3 2

0.43 0.46 - 0.64"

23.9 44

9 X 10 3 1.3 - 2.8

0.018 0.012

× 106 a Conditions: 0.05 M Tris buffer, pH 7.2, 25°C; automatic method a Values corresponding to right fits between the experimental points and the M W C model (see text)

Int. J. Biol. Macromol., 1991, Vol. 13, October

269

Covalent fixation of benzene hexacarboxylate onto human haemoolobin: M. Ldonard and E. Dellacherie O.D. 280 nm

A.1

OD

phosphate [mM]

~

280 nm

HbA 200

A2

.-'"'"

(1

100 I

I

I

I

I

t

I

I

Al A 1 1

O.D. 280 nm

2 AI

phosphates [mM]

200

c

b

..--":

1

100

Ve ml 0

100

200

300

400

Figure 6 Cation exchange chromatography (CM 52) in

sodium phosphate buffer pH 5.8: (a) of conjugate A; (b) of conjugate C (2 mol BHC-MPOE/mol Hb and 4 mol EDCI/mol BHC-MPOE) gel filtration profiles of the crude mixtures (Figure 3) and as IHP has no effect on their Pso. One can assume that certain modified Hb molecules rearranged during the cation-exchange chromatography step to give rise to more stable tetramers, as follows:

2(~tfl)*afl ~ (ctfl)* + (~fl)z (~fl)*= Hb dimer substituted with one or several BHC-MPOE molecules. This kind of rearrangement has already been proposed for Hb modified with pyridoxal phosphate 4 or adenosine triphosphate 9. The medium peak (A1 or B1) was assigned to an Hb derivative with 2 mol BHC-MPOE linked per mol Hb. In fact, the elution volume of this peak was the same as that obtained for another conjugate (conjugate C, Figure 6b) prepared from oxyHb under different conditions, i.e. still 2 mol B H C - M P O E / m o l Hb but 4 mol EDCI/mol BHC-MPOE (instead of 1). In this case, only one peak was observed and no BHC-MPOE remained in the reaction mixture, which yielded the stoichiometry of the conjugate unambiguously. The difference between the conjugates A (or B) and C results from the great amount of the condensation agent, EDCI, used in the synthesis of C which provoked intramolecular crosslinking inside the Hb molecule and avoided all kinds of rearrangement. Tryptic peptide mapping Globins of the major fractions A 1 and B~ obtained after chromatography on CM 52, were prepared by precipitation with acetone HC1 and analysed according to Clegg et al. 26. The chromatogram of A~, shown in Figure 7, exhibits four peaks, two corresponding to

270

Int. J. Biol. Macromol., 1991, Vol. 13, October

J I

]

I

I

I

0.5

I

I

I

1

0

Figure 7 Cation exchange chromatography (CM 52) of the constituent globin chains from native Hb and fraction A1 according to Clegg et al. 26. Vr is the total volume used for the gradient, i.e. 250 ml unmodified c~- and fl-globins, and two, A~ and A 2, corresponding to modified chains. The same profile was obtained for B r The percentages of modified ct- and fl-chains were evaluated by comparing the peak areas of remaining unmodified globins with those corresponding to the same amount of Hb: about 80% of fl-globins and 15% of ct-globins were modified in A1, 80% and 10% respectively in B1. The modified globins B~, B~, and A~, A~ were subjected to tryptic digestion and the tryptic peptides were first applied to reverse h.p.l.c, as described in the experimental section. The elution profiles corresponding to A~ and A 2 are shown in Figure 8. Those corresponding to B~ were identical, and are not shown. The peptide elution profile obtained from the A 2 globin shows that it is a fl-globin in which the T1 (fll-fl7) fragment is missing. As T2 is not missing, this means that T1 is modified on the ~-terminal NH 2 of Val ill. However the elution profile obtained from A~ is far less easy to identify, as all the fragments of the ~-chains can be found in it, together with fragments identified in the peptide elution profile obtained from native fl globins. This probably means that A~ results from the cross-linking of an a- and a fl-globin by means of a polymeric BHC. However the peptide elution profile is not precise enough to identify the site of cross-linking. In another experiment, the tryptic peptides corresponding to the A 2 and B 2 fractions were subjected to gel permeation chromatography (AcA 54 Ultrogel) in order to isolate the polymer-modified fragments. The results of the amino acid analysis of these high molecular weight fractions are shown in Table 3. It can be seen that amino acids not present in Tiff were found (Gly, Ala, Phe, Ser, Asp + A s n , Cys) and that the composition in Leu was very high. Taking into account the composition of various possible fl tryptic fragments and the fact that BHCM P O E possesses an affinity for the polyphosphate binding site, we assumed that a few BHC-MPOE molecules could bind to Lys fl82 and that the BHCMPOE-substituted T9-T 10 block could be found, together with modified TI. The theory which best fitted the

Covalent fixation of benzene hexacarboxylate onto human haemoglobin: M. L#onard and E. Dellacherie Table 3 Amino acid composition of polymer-modified tryptic peptides of fractions A2 and B~

Val Leu Gly Ala Phe Ser Asp +Asn His Lys Thr Glu + Gln Cys Pro

i (x

|

6O

5'0

4'0

3'0

2'0

I"0

0

b

~213

2

AI

3

m[

B2

Calculateda

1.1 2.8 0.8 1 0.8 0.5 1.3 1.2 1.6 1.2 2.35 0.4 1.2

1.2 2.4 1 0.9 0.7 0.6 0.9 1.4 1.6 1.2 2.5 0.25 1.2

1.25 2.5 0.75 0.75 0.5 0.5 1 1.4 1.5 1.4 2.25 0.25 1

a Calculated by assuming that in A~ and B 2 all the modified fl chains are substituted by B H C - M P O E on Val 1 (T1) and 25% on Lys 82 (T9-T10), and normed with respect to Pro

/ mt

A2

( 6"0

50

4'0

3"0

~"--"

'--------" ~ " 2"0 Ib-

t

0

Figure 8 C s h.p.l.c, tryptic peptide maps of the globin fractions A~(a) and A2(b). For conditions see Experimental section experimental values was that among the Val 1-substituted fl chains ofA 2 and B 2, 25% were also modified on Lys 82.

Discussion The data reported here firstly show that under defined conditions ( B H C - M P O E / H b = 2 mol/mol; EDCI/BHC-

M P O E = 1 mol/mol) the polymeric effector BHC-MPOE reacts onto oxy and deoxyHb to give rise to low oxygen affinity conjugates (A and B) with similar structural and oxygen-binding properties. Under other conditions, and specially when the amount of EDCI was increased, the results were quite different and whereas the conjugates prepared from deoxyHb still presented a high P5o, those obtained from oxyHb exhibited altered oxygen-binding properties. For example conjugate C, prepared from oxyHb with 2mol B H C - M P O E / m o l Hb and 4mol E D C I / m o l B H C - M P O E exhibited a Pso of only 11 Torr (0.05 M Tris buffer, pH 7.2, 25°C). The functional properties of the two kinds of conjugates, A and B (prepared from oxy and deoxyHb respectively), are qualitatively similar to those of 2,3-DPG-saturated Hb or of certain chemically modified Hbs 11"14. Pso is greatly increased (Table 1), cooperativity is maintained ( Table 2) and the Bohr effect is present although reduced (Figure 5). The increase in Pso can be accounted for on the basis of an increase both in K T and K R, which indicates that the T state is overstabilized (phosphate-like mechanism) and that the R conformation has a reduced affinity for oxygen. The cooperativity curve is highly asymmetric (Figure 4b) as a significant cooperativity is retained at Y--0.97, while the cooperativity almost vanishes at Y = 0.1, as is classically observed when I H P is added to Hb 3°. All these characteristics, together with the fact that firstly IHP has no effect on the oxygen affinity of the conjugates, and, secondly, the reaction of B H C - M P O E onto Hb is greatly hampered when IHP is present, suggest that B H C - M P O E preferentially reacts inside the polyphosphate-binding site. This selectivity could be the result of a specific binding, followed by covalent bond formation. The peptide mapping of the two conjugates prepared from oxy- and deoxyHb confirms this hypothesis. It firstly shows that for both conjugates the major constituent is identical, composed of Hb molecules substituted by 2 mol of B H C - M P O E on average. In this fraction, 80% of fl-globins and only 10-15% of ct-globins are modified. The peptide mapping of the modified ~ and fl globins are evidence that all the modified or-chains were in fact crosslinked to fl-chains by means of a polymer-linked BHC and that all the other modified fl globins were

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271

C o v a l e n t f i x a t i o n o f b e n z e n e h e x a c a r b o x y l a t e o n t o h u m a n h a e m o g l o b i n : M . I A o n a r d a n d E. D e l l a c h e r i e

substituted on their Val 1 residues, 25% a m o n g them being also substituted on Lys 82. Thus, the preferential fixation of B H C - M P O E o n t o H b fl-cleft amino acids explains the oxygen-binding properties of the resulting conjugates, the partial fixation onto Lys fl82 giving a possible explanation for the reduced Bohr effect. The fact that the derivatives A and B exhibited the same structural and functional properties whatever the ligation state of H b during the reaction, is rather unusual, specially with a binding to Val El. We had assumed that in the reaction onto oxyHb, the Val ~1 residue could not be modified because of steric hindrance connected with the size of the polymeric moiety of B H C - M P O E . However, when unmodified B H C was allowed to react with oxyHb, low oxygen affinity derivatives were also obtained. This probably means that B H C strongly interacts with the E-cleft even in the o x y H b as already shown with 2 , 3 - D P G 31. Finally, with regard to transfusional purposes, such polymeric conjugates, easily prepared from oxyHb, could be of a great interest. Thus, conjugate A can unload 48% of its oxygen between 100 and 40 Torr at 25°C, p H 7.2, against 40% for blood. Other conjugates prepared from dextran-linked B H C or benzene tetracarboxylate and o x y H b were found to exhibit similar oxygen-binding properties 32 probably because of the same specific fixation to fl cavity amino acids. M o r e o v e r these products, as well as those obtained after treatment in 1 M MgC12 according to Benesch and K w o n g 13, were found to possess molecular weights high enough to be possibly retained inside the somatic capillary beds. I n vivo experiments on rats are now under way in order to assess the potential ability of these polymer-modified Hbs to carry satisfactorily oxygen in transfusional fluids.

Acknowledgements We are grateful to Dr C. Poyart for his helpful advice in the study of the Hb functional properties and to J. Kister for his assistance in the computational aspects of this study. We thank Dr H. Wajcman for his help in the peptide mapping.

References 1 Benesch, R., Benesch, R. E., Renthal, R. D. and Laeda, N. Biochemistry 1972, 11, 3576

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