©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A Doubly Cross-linked Human Hemoglobin
EFFECTS OF CROSS-LINKS BETWEEN DIFFERENT SUBUNITS (*)

(Received for publication, July 17, 1995; and in revised form, October 25, 1995)

Richard T. Jones (1) (2) Daniel T. Shih (1) Thomas S. Fujita (1) Yonghong Song Hong Xiao (1) Charlotte Head (1) Ronald Kluger (2)

From the  (1)Department of Biochemistry and Molecular Biology, School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201 and (2)Lash Miller Chemical Laboratories, Department of Chemistry, University of Toronto, Toronto, Canada M5S 1A1

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human deoxyhemoglobin cross-linked with trimesyl tris(3,5-dibromosalicylate) produces the previously reported cross-linked hemoglobin in which the amino groups of the two beta chain 82 lysyl residues are joined by a trimesyl bridge. Further specific modification of this protein directed to the alpha subunits with bis(3,5-dibromosalicyl)fumarate gives a doubly cross-linked material in which the -amino groups of the two alpha chain 99 lysyl residues are now joined by a fumaryl bridge. The singly cross-linked beta chain species binds oxygen cooperatively with a high oxygen affinity (P = 4.8 torr at pH 7.4). The addition of the second cross-linking reduces the oxygen affinity to 15.9 torr, which compares with 13.0 torr for the singly cross-linked alpha chain species. The doubly cross-linked hemoglobin retains significant cooperativity with a Hill coefficient of 2.3 compared with 3.0 for unmodified hemoglobin. Because some of the groups responsible for the Bohr effect are acylated, this doubly cross-linked hemoglobin exhibits 25% of the normal Bohr effect and less than 20% of the normal chloride effect. The use of two distinct cross-links within the same tetramer provides a material for physical and structural analysis as well as for further modifications for specific applications. The results indicate that the cross-link introducing the lowest oxygen affinity in the two singly cross-linked species appears to control the overall affinity in this doubly cross-linked species.


INTRODUCTION

Specifically cross-linked hemoglobins are useful for the investigation of structure-function relationships, (Schumacher et al., 1995) as well as in the development of potential substitutes for red blood cell transfusion (Snyder et al., 1987; Keipert et al., 1989; Bucci et al., 1989; Vandegriff and Winslow, 1991; Kluger et al., 1994). By retaining a reactive site in a cross-linker after modification of hemoglobin, other modifications can be readily prepared by addition of nucleophiles to the modified hemoglobin (Kluger and Song, 1994). This makes it possible to use hemoglobin as a carrier for drugs or other chemicals that may have pharmacologic value (Seymour, 1992) or provide probes for physical and structural analysis. However, hemoglobin containing a trimesyl dibromosalicylate ester as the bisamide of the -amino groups of both beta82 lysine residues (beta82-TDBS-82beta; (^1)schematic structure is shown in Fig. Z1), has a relatively high affinity for oxygen.


Figure Z1: Structure 1



Due to its high affinity for oxygen, this is unlikely to deliver oxygen efficiently. Since combining oxygen delivery and bioconjugation is potentially of theoretical as well as medicinal significance, we considered the possibility of further modification to cause a reduced oxygen affinity by introducing a second cross-link.

Walder et al.(1994) developed an efficient hemoglobin-based oxygen carrier that contains a fumaryl bisamide of the -amino groups of both alpha99 lysine residues (alpha99-F-99alphabeta(2); the Baxter Healthcare red cell substitute-derived form of this is called DCLHB) (Walder et al., 1994; Chatterjee et al. 1986; Snyder et al., 1987). If the fumaryl cross-link at the alpha99 lysines is added to alpha(2)beta82-TDBS-82beta (Kluger et al., 1992b), the oxygen affinity might be altered to make it suitable as an oxygen carrier (schematic structure shown in Fig. Z2).


Figure Z2: Structure 2



Whatever the case, the structure and properties of the doubly cross-linked material would provide an interesting basis for functional studies. We have been able to produce these chemical modifications of the same hemoglobin efficiently. The resulting material has the desirable features of both individual modifications. Olsen et al.(1991) have also reported in abstract form the preparation of multilinked hemoglobins.


MATERIALS AND METHODS

Hemoglobin Preparation and Modification

Hemoglobin solutions were prepared as described previously (Jones et al., 1994) and stored as COHb at 0 °C on ice. For reaction with TTDS, COHb (0.5 mM in pH 9.0, 0.05 M sodium borate) was converted to deoxy-Hb by photoirradiation under a stream of humidified oxygen for 60 min at 0 °C in a rotating flask (Shih et al., 1982). Oxygen was removed by passing a steam of humidified N(2) over the oxy-Hb solution for 1.5 h at 35 °C in the rotating flask. For analytical studies, reactions were carried out with 100 mg of protein while preparative studies were conducted on 1-2-g samples.

TTDS was synthesized and analyzed as previously reported (Kluger et al. 1992b). Because TTDS is not readily soluble in water, 1,4-dioxane was used to dissolve the reagent. (We have also added the reagent in water with sonication.) The solution of TTDS was degassed under an aspirator and infused with nitrogen to remove oxygen. An amount of this solution was added to the deoxy-Hb to make the final concentrations of Hb 0.5 mM and TTDS 0.75 or 1.0 mM. The reaction was allowed to proceed for up to 2 h under a stream of humidified N(2) at 35 °C in the same rotating flask as used for the deoxygenation.

DBSF was either synthesized as described by Walder et al. (1979) or purchased. Although DBSF can be added as a solid to deoxy-Hb (Walder et al., 1994), we added it as a solution in 1,4-dioxane and 0.1 M pH 9.0 borate. (We have also added the reagent in water with sonication). Oxygen was removed in the same way for TTDS solutions. An amount of DBSF was added to the deoxy-Hb that had been reacted with TTDS to equal twice the moles of Hb present, i.e. to make the DBSF about 1.0 mM. This reaction was allowed to proceed for another 2 h under a stream of humidified nitrogen at 35 °C in the same rotating flask as used for the reaction with TTDS. The final reaction mixture was then cooled to 0 °C in an ice bath, and the modified Hb was converted to the CO form by passing a stream of humidified carbon monoxide over the solution for 2-3 min. The COHb mixture was separated from the excess reagents and low molecular weight reaction products by passing through a Sephadex G-25 column equilibrated with 0.005 M pH 8.0 MOPS and concentrated by pressure filtration. Portions of the stripped COHb were stored on ice, while others were flash-frozen with dry ice and stored at -80 °C until purified or studied further.

Preparative Example

Twenty ml of 0.1 M borate, pH 9.0, was combined with 1.44 g of COHb in 18 ml of water and oxygenated to remove the CO and then deoxygenated. To this was added 37 mg of TTDS in 7 ml containing 2.5 ml of dioxane, 2.5 ml of 0.1 M pH 9.0 borate buffer, and 2.0 ml of water. After 1.5 h of reaction, 30 mg of DBSF in 3 ml of solution containing 1.0 ml of dioxane, 1.5 ml of 0.1 M pH 9.0 borate, and 0.5 ml of water was added to the reaction mixture under nitrogen. After 2 h the reaction was cooled in ice, converted to COHb, and passed though the gel filtration column.

Characterization of Modified Hemoglobins

The procedures for analytical and preparative separations of hemoglobins by ion exchange chromatography, globin chain separation by reversed phase HPLC on C-4 columns, enzyme hydrolysis with trypsin and Glu-C endoproteinase, and peptide and amino acid analysis are described in detail elsewhere (Jones, 1994). The molecular masses of globin chains were measured by electrospray ionization mass spectrometry (Fenn et al., 1989).

Measurement of Functional Properties of Isolated Hemoglobins

The oxygen binding equilibria of modified hemoglobins were measured by the automatic recording method of Imai et al.(1970) using the apparatus and procedure described by Shih and Jones(1986). The conditions for comparing the oxygen affinities of the modified hemoglobins were 50 mM Bis-Tris, pH 7.4, 0.1 M Cl, 25 °C, and 55 µM heme. The parameters measured were the oxygen pressure for half-saturation (P) and Hill's coefficient of cooperativity at half saturation (n). The Bohr (H) effect was measured between pH 6.0 and 9.0 and calculated for the interval of pH 7.0-8.0. The chloride effect was measured for 0.007-0.5 M NaCl.

Rates of Hydrolysis of DBS from alpha(2)beta82-TDBS-82beta and alpha99-F-99alphabeta82-TDBS-82beta

The rates of hydrolysis of DBS from alpha(2)beta82-TDBS-82beta and betaalpha99-F-99alphabeta82-TDBS-82 with Hbs at 0.1 mM were followed at pH 5.5 in 0.1 M citrate, pH 7.2 in MOPS, and pH 9.0 in borate at 45 °C by measuring the amounts of beta82-TDBS-82beta and beta82-T-82beta chains relative to total alpha chains present at various times by C-4 globin chain separation. Samples of modified hemoglobin were removed from the hydrolysis solutions and flash-frozen with dry ice in order to stop the hydrolysis reaction until the C-4 reversed phase HPLC could be done. The rates of hydrolysis were estimated from the rate of decrease of beta82-TDBS-82beta as well as the rate of increase of beta82-T-82beta.


RESULTS

The reaction of deoxy-Hb with two equivalents of TTDS in borate buffer at pH 9 (producing alpha(2)beta82-TDBS-82beta) followed by reaction with two equivalents of DBSF yields the doubly cross-linked hemoglobin tetramer containing an intact ester linkage (i.e. two cross-links connecting four lysine side chains with a 3,5-dibromosalicylate ester on the trimesyl betabeta-cross-link), alpha99-F-99alphabeta82-TDBS-82beta. The structure is shown schematically in Fig. Z3.


Figure Z3: Structure 3



Influence of Reaction Conditions on Extent of Product Formation

Conditions for optimal formation of this doubly cross-linked product were determined through trials under varying conditions. We have shown that the extent of reaction of TTDS with hemoglobin is greater with deoxy-Hb than with COHb at 35 °C (0.1 M pH 7.2 MOPS) (Kluger et al., 1992b). In the present study, three other reaction conditions were tested: 0.1 M pH 7.2 bis-Tris, 0.1 M pH 8.0 MOPS, and 0.05 M pH 9.0 borate. The greatest conversion of Hb A to alpha(2)beta82-TDBS-82beta was obtained using 0.05 M pH 9.0 borate. Fig. 1shows the results of a typical C-4 reversed phase HPLC globin chain separation of the Hb mixture after 0.5 h of reacting 0.5 mM deoxy-Hb with 0.75 mM TTDS in 0.05 M pH 9.0 borate at 35 °C. Different conditions were also tried for the second reaction with DBSF; however, optimal results were obtained with 1.0 mM DBSF under the same conditions as the first reaction, i.e. 0.5 mM deoxy-Hb, 0.05 M borate, pH 9.0, 35 °C. Fig. 2shows the globin chain separation of a typical preparation of the doubly cross-linked Hb. From both Fig. 1and Fig. 2it is apparent that partial hydrolysis of DBS from the beta82-TDBS-82beta chains occurs under the preparation conditions. This can be decreased by reducing the temperature of the reaction and increased by raising the reaction temperature. Eighty percent or more conversion of beta82-TDBS-82beta to beta82-T-82beta was observed when the reaction mixture was converted to CO and then heated to 60 °C for 3 h after an initial 1 h for reaction with DBSF at 35 °C as shown in Fig. 3. More than 95% conversion has been observed after storing a stripped reaction mixture for 2 months in the cold room at 4 °C. No hydrolysis was observed when alpha(2)beta82-TDBS-82beta was stored at -80 °C.


Figure 1: Globin chain separation on C-4 reversed phase column after reaction of 0.5 mM deoxy-Hb with 1.0 mM TTDS in 0.05 M borate at pH 9.0 and 35 °C for 30 min. More than 95% of the beta chains were chemically modified mostly to beta82-TDBS-82beta (peak eluting at 55 min).




Figure 2: Globin chain separation on C-4 reversed phase column after reaction of 0.5 mM deoxy-Hb in 0.05 M borate at pH 9.0 and 35 °C with 1.0 mM TTDS for 1 h. followed by 1.0 mM DBBS for another 1 h. All of the beta chains were chemically modified mostly to beta82-TDBS-82beta and its hydrolysis product beta82-T-82beta. In this preparation about 85% of the alpha chains were chemically modified to alpha99-F-99alpha, and 15% remained unmodified.




Figure 3: Globin chain separation on C-4 reversed phase column of the same reaction mixture shown in Fig. 2after heating to 60 °C for 3 h. The primary change is the conversion of all but about 20% of the beta82-TDBS-82beta mainly to beta82-T-82beta.



Kluger and Song(1994) have reported that alpha(2)beta82-TDBS-82beta will react with a variety of nucleophiles to form derivatives with the third carboxyl of the trimesyl cross-linker. Fig. 4shows the chain separation results after reacting a sample of stripped reaction mixture containing alpha99-F-99alphabeta82-TDBS-82beta with 0.07 M lysyl-lysine at 35 °C in 0.05 M borate, pH 9.0, for 2 h. Other nucleophiles including glycine, lysine, and Tris react under the same conditions to convert the alpha(2)beta82-TDBS-82beta to covalently linked derivatives.


Figure 4: Globin chain separation on C-4 reversed phase column of a similar reaction mixture shown in Fig. 2after treating it with 0.07 M lysyl-lysine for 2 h more at 35 °C. The globin peak eluting at about 40 min was the conjugate of lysyl-lysine with beta82-T-82beta.



Structural Characterization of Modified Globin Chains

A reaction mixture that contained some unreacted Hb A and some partially hydrolyzed alpha99-F-99alphabeta82-TDBS-82beta was separated with a C-4 reversed phase HPLC column interfaced to an electrospray ionization mass spectrometer. The molecular masses of the material eluting in the positions of unmodified beta and alpha chains as well as peaks believed to be beta82-T-82beta, beta82-TDBS-82beta, and alpha99-F-99alpha chains were determined and are listed in Table 1. The observed molecular masses are coincident with the calculated values within the experimental uncertainty limits of the method.



The peaks assigned to the beta82-T-82beta and alpha99-F-99alpha globin chains were isolated by preparative C-4 reverse phase HPLC for further structural characterization. The beta82-T-82beta chains were hydrolyzed to a set of peptides with trypsin followed by Glu-C endoproteinase. The alpha99-F-99alpha chains were oxidized with performic acid and then hydrolyzed with trypsin in the presence of 2 M urea. Fig. 5, A and B, show the C-18 reverse phase HPLC peptide pattern of these globin chains. In Fig. 5A, all of the normal tryptic, Glu-C peptides of the beta chain are present except for those that occur from tryptic cleavage adjacent to Lys: betaT-9 and betaT-10a. A tryptic peptide not found in native Hb elutes at about 94 min and absorbs at 258 nm, indicative of the presence of trimesyl moiety. Amino acid analysis indicates it has the aminoacyl components of betaT-9,10a. These results are the same as those reported earlier for alpha(2)beta82-T-82beta obtained from the reaction of COHb with trimesoyl tris(methylphosphate); Kluger et al., 1992a). This peptide pattern and the amino acid composition of the new peptide combined with the molecular mass measurement of the globin chain are the basis for deducing the structure to be beta82-T-82beta. Similarly, Fig. 5B shows all of the normal tryptic peptides observed for oxidized alpha chains except for the absence of alphaT-11 and oxalphaT-12. In their place is a new peptide that elutes near the end of the chromatogram that has the amino acid composition of oxalphaT-11,12. These results are the same as those found for the alpha99-F-99alpha present in a preparation of DCLHB from Baxter Healthcare Corp. and reported elsewhere (Jones, 1994).


Figure 5: A, HPLC peptide pattern of a trypsin-Glu-C hydrolysate of globin corresponding to the material eluting at about 45 min from the C-4 column separations (see Fig. 1Fig. 2Fig. 3Fig. 4). The peptides were separated on a C-18 reversed-phase column using a water-acetonitrile gradient in 0.1% trifluoroacetic acid. All of the normal peptides were present except betaT-9 and betaT-10a. A new peptide was found eluting at about 94 min, which had the amino acid composition of betaT-9 and betaT-10a plus the UV absorption of the trimesic cross-linker. B, HPLC peptide pattern of a trypsin hydrolysate of the oxidized globin corresponding to the material eluting at about 75-77 min from the C-4 column separations (see Fig. 2and Fig. 3). The peptides were separated as described for panel A. All of the normal peptides were present except alphaT-11 and alphaT-12. A new peptide was found eluting at about 83 min. which had the amino acid composition of alphaT-11 and alphaT-12.



Rates of Hydrolysis of DBS from alpha(2)beta82-TDBS-82beta and alpha99-F-99alphabeta82-TDBS-82beta

Table 2lists the rate constants of hydrolysis of the ester moiety as followed by the decrease in the relative amount of beta82-TDBS-82beta and the increase of beta82-T-82beta chains (by HPLC analysis) in samples containing either single or doubly cross-linked Hbs. The data accurately fit integrated first order rate expressions (collected over at least two half-times). The rate of hydrolysis increases with pH but with a slope of less than unity, indicating a change from an uncatalyzed to base-catalyzed mechanism occurs over the pH range studied. Most of the kinetic studies we report were done using alpha(2)beta82-TDBS-82beta because of the ease of estimating the amounts of beta82-TDBS-82beta and beta82-T-82beta relative to unmodified alpha chains. We find comparable hydrolysis rates of DBS in the singly and doubly cross-linked Hb under the same conditions at pH 7.2.



Functional Properties of the Modified Hemoglobins

The oxygen-binding properties of alpha99-F-99alphabeta82-T-82beta were measured and compared with those of unmodified Hb A, alpha99-F-99alphabeta(2), and alpha(2)beta82-T-82beta and are listed in Table 3. The P of alpha99-F-99alphabeta82-T-82beta is about three times larger than that of unmodified Hb A at pH 7.4 but comparable with that of alpha99-F-99alphabeta(2), which has potential as a blood substitute (Chatterjee et al. 1986).



A trimesyl cross-link between the two Lys residues does not appear to change P appreciably from that of unmodified Hb at this pH. Differences in the Bohr effects do result in different outcomes at other pH levels. It is likely that there is little difference in structure of this species compared with native Hb in the oxy or deoxy forms. However, a fumaryl cross-link between the two Lys residues increases P by a factor of 2.5, indicating a marked decrease in oxygen affinity through a higher energy R state, lower energy T state, or a combination of both. Thus, the two types of cross-links in this double cross-linked Hb are manifested very differently in their effects on oxygen affinity. The Hill coefficients at pH 7.4 are somewhat reduced compared with that of unmodified Hb for both alpha(2)beta82-T-82beta and alpha99-F-99alphabeta82-T-82beta with n values of 2.4 and 2.3, respectively, while n = 2.7 for alpha99-F-99alphabeta82-T-82beta at pH 7.0. (^2)Both the Bohr effect and chloride effect are lower than the normal values for unmodified Hb (in decreasing order: alpha(2)beta82-T-82beta to alpha99-F-99alphabeta(2) to alpha99-F-99alphabeta82-T-82beta).


DISCUSSION

The measurement and interpretation of the effects of one or two cross-links on the properties of hemoglobin depends on being able to produce these species efficiently. The reaction of TTDS with hemoglobin is highly selective for the -amino groups of the Lys residues in the DPG binding site of the central cavity of hemoglobin (Kluger et al., 1992b). The yield of modified hemoglobins is over 95%, the products being mainly either alpha(2)beta82-TDBS-82beta or alpha(2)beta82-T-82betaalpha(2), when the preparation is carried out under nitrogen (pH 9.0 in borate, 35 °C), even with only a 50% molar excess of TTDS over Hb. The relative amount of alpha(2)beta82-T-82beta compared with alpha(2)beta82-TDBS-82beta at the end of the reaction depends upon the extent of hydrolysis of DBS from the third carboxyl group of the trimesic cross-linker, which in turn depends upon the temperature, pH, and duration of the reaction.

Directing the Second Reagent to the alpha Subunits

The introduction of a second cross-link by reaction between amino groups of the alpha subunits proceeds with high efficiency in the species containing the cross-link between beta subunits. The conversion of the unmodified alpha chains of the hemoglobins in the reaction mixture resulting from treatment of deoxy-Hb with TTDS to cross-linked alpha chains by reacting the mixture with DBSF under nitrogen is about 85% complete at pH 9.0 and 35 °C. Consistent with expectations, this yield is significantly greater than for the cross-linking of only alpha chains of deoxy-Hb using DBSF in the presence of inositol hexaphosphate (to direct DBSF away from the DPG binding site) under the most favorable conditions reported by Walder et al.(1994). Although these authors state that ``the yield of this derivative is markedly increased in the presence of polyanions that bind within the DPG site'' they do not report the extent of reaction. Using their conditions, we have been able to obtain a maximum of about 55% yield of alpha99-F-99alphabeta(2) compared with the starting amount of unmodified Hb.^2 The association of inositol hexaphosphate with Hb is a dynamic process involving association and dissociation, so that the competition remains between the reagents for the site. On the other hand, covalently blocking the DPG site involves no dissociation, permanently directing reagents toward other sites. Thus efficient covalent modification directs the reaction of DBSF to the -amino groups of the Lys residues.

By heating the final reaction mixture used to produce the doubly cross-linked species to 60 °C for 3 h in the presence of CO, most of the DBS will be hydrolyzed from the trimesic cross-linker, and the main product is alpha99-F-99alphabeta82-T-82. This approach to the preparation of a modified Hb with an alpha99-F-99alpha cross-link may be an efficient alternative to the use of polyanions to promote formation of this modification.

Structural Analysis

The identification of the cross-link sites for alpha99-F-99alphabeta82-T-82beta and alpha99-F-99alphabeta82-TDBS-82beta is based on analysis of the various types of globin chains isolated from the reaction mixtures and purified Hb components, their molecular masses, and their peptide patterns. The globin chain peak that elutes at about 46 min from the C-4 column had a molecular mass of 31,908, in agreement with the calculated mass of 31,910 for 2beta chains with one molecule of trimesic acid attached as the bisamide (see Fig. 2and Table 1) The peptide pattern of the material after enzymatic hydrolysis showed it to be beta chains with the Lys groups blocked and cross-linked by the trimesyl group. Therefore, this is beta82-T-82beta. The globin chain peak that elutes at about 57 min has an observed mass of 32,185 in agreement with the calculated mass of 32,188 for 2beta chains with one molecule of trimesyl bisamide and as a DBS ester. Upon standing at 0 °C for several weeks or after heating, this globin chain elutes in the position of beta82-T-82beta with an equimolar amount of DBS eluting just ahead of the heme from the C-4 column. Thus, this modified globin is beta82-TDBS-82beta, i.e. the same as beta82-T-82beta but with the third DBS still present as an ester of the third carboxyl group of the trimesic acid cross-linker. The globin chain peak that elutes at about 76 min has an observed mass of 30,335 in agreement with the calculated mass of 30,333 for 2alpha chains connected as a fumaryl bisamide. Its peptide pattern shows this modified globin to contain alpha chains with the Lys residues blocked by the fumaryl cross-link. Therefore, it is alpha99-F-99alpha. The identification of the type of globin chains present in the purified hemoglobin fractions permits us to deduce with confidence the cross-linked structures to be alpha(2)beta82-T-82beta, alpha(2)beta82-TDBS-82beta, alpha99-F-99alphabeta(2), alpha99-F-99alphabeta82-T-82beta, and alpha99-F-99alphabeta82-TDBS-82beta.

As Kluger and Song(1994) observed for alpha(2)beta82-TDBS-82beta, we find that alpha99-F-99alphabeta82-TDBS-82beta can act as an acylating reagent toward nucleophiles, forming conjugates of hemoglobin. This presents opportunities to investigate combining oxygen delivery with bioconjugation for a transfused or perfused material. It also permits the attachment of probes and other molecules that may be useful in physical and structural analyses.

Ester Hydrolysis and Environment

The rate of hydrolysis of the dibromosalicylate ester of the cross-linker on the both cross-linked proteins described above, compared with the rate of hydrolysis of unreacted TTDS in solution gives an indication of the accessibility of the ester. The observed first order rate coefficient for hydrolysis of DBS from both alpha99-F-99alphabeta82-TDBS-82beta and from alpha(2)beta82-TDBS-82beta (at 35 °C in 0.1 M phosphate buffer; Table 2) is 7 times 10 s. The rate coefficient for hydrolysis of the first dibromosalicylate ester of TTDS in 0.1 M MOPS buffer is 3.2 times 10 s, comparable with the rate for the protein derivative. Thus, from this comparison we conclude that in the protein derivatives, the ester is not protected from hydrolysis. This suggests that the ester group is extended toward the solvent rather than into the DPG binding pocket of the protein. We are attempting to obtain crystals of the protein with the intact ester so that the structure may be analyzed by x-ray diffraction.

In alkaline solutions, the rate of hydrolysis of DBS from beta82-TDBS-82beta increases. Thus, by controlling the pH of the solution in which the product is kept, the free acid alpha99-F-99alphabeta82-T-82beta or the ester, alpha99-F-99alphabeta82-TDBS-82beta, will predominate. The latter is useful for subsequent reaction with nucleophiles that form stable derivatives by displacement of the ester retained on the trimesyl cross-link.

Relationship of Oxygen Binding and Cross-links

Comparisons of the P values of alpha(2)beta82-T-82beta, alpha99-F-99alphabeta(2), and alpha99-F-99alphabeta82-T-82beta indicate that the alpha-99 fumaryl cross-linkage has the dominant effect on the oxygen affinity of the doubly cross-linked Hb. Although the P of alpha(2)beta82-T-82beta is similar to that of unmodified Hb A at pH 7.4, the decreases in its cooperativity (n), Bohr and chloride effects show that this structural modification significantly changes its functional properties from those of Hb A. Therefore, both the cross-linking of the beta chains with the trimesyl bridge as well as the cross-linking of the alpha chains with the fumaryl bridge contribute to the changes in the overall oxygen binding properties of this doubly cross-linked Hb.

It is well documented that nearly 45% of the overall alkaline Bohr effect is due to the releasing of protons from the His residues in human Hb A that occurs with the decrease in pK associated with oxygenation (Kilmartin et al., 1980; Shih et al., 1993). The remaining 55% of the Bohr effect is attributed to the release of protons linked to the chloride effect. The positively charged cluster of amino acid residues in the central cavity, i.e. alpha-NH(2) of Val, -NH(2) of Lys and Lys, and Arg, has been identified as important in the structural mechanism of the chloride effect (Perutz et al., 1994). According to Perutz et al.(1994), modifications of any positively charged residue in the central cavity should change the chloride effect and thus the Bohr effect. Therefore, the additive losses of Bohr effect and chloride effect in the order of alpha(2)beta82-T-82beta, alpha99-F-99alphabeta(2), and alpha99-F-99alphabeta82-T-82beta would be expected because of the extent of acylation of the -NH(2) groups of Lys and Lys. Our data show that the cross-linking modification of Lys reduces the chloride effect by 55% and the cross-linking of Lys reduces it by 66%. The accompanied decrease in overall Bohr effect with each modification is 26 and 48%, respectively. In the case of the doubly cross-linked alpha99-F-99alphabeta82-T-82beta Hb in which four of the positively charged residues in the central cavity have been eliminated, the accumulated reduction of chloride effect of 84% and accompanying large reduction of Bohr effect of 73% are consistent with the concept of chloride binding proposed by Perutz et al.(1994).

As noted above, His is known to be responsible for the chloride-independent part of the overall alkaline Bohr effect of Hb A. Although the double cross-linking modifications do not chemically alter the histidyl residues of beta146, the reduction of the Bohr effect of this hemoglobin to 27% of normal, which is about one-half of the chloride-independent part of the Bohr effect, is probably due to constraints in oxygen-linked structural changes in the doubly cross-linked Hb. The high P and low n values of the doubly cross-linked Hb, which indicate a reduction in the shift in the allosteric equilibrium toward the R state, support this explanation. The extra structural constraint in the alpha99-F-99alphabeta82-T-82beta Hb that is manifest in its decreased cooperativity is presumably due more to the cross-linking modification of the two Lys residues than the Lys residues because the modification of the latter two does not influence the n value of alpha99-F-99alphabeta(2) as shown in Table 3.

These results show that sequentially introduced site-specific cross-links provide practical means for adjusting physical properties of proteins. The use of a reactive ester on one of the cross-links permits the introduction of probes for further analysis, conjugation, or modification.


FOOTNOTES

*
Work at the Oregon Health Sciences University was supported by Grant HL20142 from the Heart, Lung, and Blood Institute of the National Institutes of Health. Work at the University of Toronto was supported by the Natural Sciences and Engineering Research Council of Canada and the Protein Engineering Network of Centres of Excellence. A preliminary report of this work was presented at the XI Congress of the International Society For Artificial Cells, Blood Substitutes, and Immobilization Biotechnology in Boston, Massachusetts, July 27, 1994. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

()
To whom correspondence may be addressed.

(^1)
The abbreviations used are: alpha(2)beta82-TDBS-82beta, Hb containing a trimesyl bisamide of the -amino groups of both Lys residues as the 3,5-dibromosalicylate; alpha(2)beta82-T-82beta, Hb containing a trimesyl bisamide of the -amino groups of both Lys residues; alpha99-F-99alphabeta(2), Hb containing a fumaryl bisamide of the -amino groups of both Lys residues; alpha99-F-99alphabeta82-T-82beta, Hb containing a fumaryl bisamide of the -amino groups of both Lys residues and a trimesyl bisamide of the -amino groups of both Lys residues; alpha99-F-99alphabeta82-TDBS-82beta, Hb containing a fumaryl bisamide of the -amino groups of both Lys and a trimesyl bisamide of the -amino groups of both Lys residues as the 3,5-dibromosalicylate; DBSF, bis(3,5-dibromosalicyl)fumarate; bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; DCLHB, diaspirin-cross-linked hemoglobin, another designation for alpha99-F-99alphabeta(2); COHb, carbonmonoxy Hb; DBS, 3,5-dibromosalicylate; DPG, 2,3-diphosphoglycerate; HPLC, high performance liquid chromatography; MOPS, 3-(N-morpholino)propanesulfonic acid; n, Hill coefficient at PO(2) = P; P, oxygen pressure (torr) where Hb is 50% oxygenated; TDBS, trimesyl mono(3,5-dibromosalicylate); TTDS, trimesyl tris(3,5-dibromosalicylate).

(^2)
R. T. Jones, D. T. Shih, T. S. Fujita, Y. Song, H. Xiao, C. Head, and R. Kluger, unpublished results.


ACKNOWLEDGEMENTS

We thank Jeffrey Weissberger, C-W., Wong, and J. Wodzinska for assistance. Ion spray mass spectra were obtained by Mary Cheung and Henrianna Pang.


REFERENCES

  1. Bucci, E., Razynska, A., Urbaitis, B. & Fronticelli, C. (1989) J. Biol. Chem. 264, 6191-9937 [Abstract/Free Full Text]
  2. Chatterjee, R., Welty, E. V., Walder, R. Y., Pruitt, S. L., Rogers, P. H., Arnone, A. & Walder, J. A. (1986) J. Biol. Chem. 261, 9929-9937 [Abstract/Free Full Text]
  3. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F. & Whitehouse, C. M. (1989) Science 246, 64-70 [Medline] [Order article via Infotrieve]
  4. Imai, K., Morimoto, H., Kotani, M., Watari, H., Hirata, W. & Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196 [Medline] [Order article via Infotrieve]
  5. Jones, R. T., Head, C. G., Fujita, T., Shih, D. T.-b., Wodzinska, J. & Kluger, R. (1993) Biochemistry 32, 215-223 [Medline] [Order article via Infotrieve]
  6. Jones, R. T. (1994) Methods Enzymol. 231, 322-343 [Medline] [Order article via Infotrieve]
  7. Keipert, P. E., Adeniron, A. J., Kwong, S. & Benesch, R. E. (1989) Transfusion 29, 768-773 [Medline] [Order article via Infotrieve]
  8. Kilmartin, J. V., Fogg, J. H. & Perutz, M. F. (1980) Biochemistry 19, 3189-3193 [Medline] [Order article via Infotrieve]
  9. Kluger, R. & Song, Y. (1994) J. Org. Chem. 59, 733-736
  10. Kluger, R., Wodzinska, J., Jones, R. T., Head, C., Fujita, T. S. & Shih, D. T.-b. (1992a) Biochemistry 31, 7551-7559 [Medline] [Order article via Infotrieve]
  11. Kluger, R., Song, Y., Wodzinska, J., Head, C., Fujita, T. & Jones, R. T. (1992b) J. Am. Chem. Soc. 114, 9275-9279
  12. Kluger, R., Jones, R. T. & Shih, D. T.-b. (1994) Art. Cells. Blood Subs. and Immob. Biotech. 22, 415-428 [Medline] [Order article via Infotrieve]
  13. Olsen, K. W., Yang, T., Zhang, Q.-Y., Huang, H. Wojnicki, S. M., Corso, T. D., Ranum, D., Dave, S., White, F. L., Soneji, V. & Zabaneh, S. (1991) Biomaterials, Artif. Cells, Immob. Biotech. 19, 460
  14. Perutz, M. F., Shih, D. T.-b. & Williamson, D. (1994) J. Mol. Biol. 239, 555-560 [CrossRef][Medline] [Order article via Infotrieve]
  15. Schumacher, M. S., Dixon, M. M., Kluger, R., Jones, R. T. & Brennan, R. G. (1995) Nature 375, 84-87 [CrossRef][Medline] [Order article via Infotrieve]
  16. Seymour, L. W. (1992) Crit. Rev. Ther. Drug Carr. Sys. 9, 135-187
  17. Shih, D. T.-b., Jones, R. T. & Johnson, C. S. (1982) Hemoglobin 6, 153-167 [Medline] [Order article via Infotrieve]
  18. Shih, D. T.-b. & Jones, R. T. (1986) Methods Hematol. 15, 124-141
  19. Shih, D. T.-b., Luisi, B. F., Miyazaki, G., Perutz, M. F., Nagai, K. (1993) J. Mol. Biol. 230, 1291-1296 [CrossRef][Medline] [Order article via Infotrieve]
  20. Snyder, S. R., Welty, E. V., Walder, R. Y., Williams, L. A. & Walder, J. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7280-7284 [Abstract]
  21. Vandegriff, K. D. & Winslow, R. M. (1991) Chemistry and Industry 497-504
  22. Walder, J. A., Zaugg, R. H., Walder, R. Y., Steele, J. M., and Klotz, I. M. (1979) Biochemistry 18, 4265-4270 [Medline] [Order article via Infotrieve]
  23. Walder, R. Y., Andracki, M. E., and Walder, J. A. (1994) Methods Enzymol. 2331, 274-280 [CrossRef]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.