Báo cáo khoa học: The oxidative effect of bacterial lipopolysaccharide on native and cross-linked human hemoglobin as a function of the structure of the lipopolysaccharide A comparison of the effects of smooth and rough lipopolysaccharide ppt

The oxidative effect of bacterial lipopolysaccharide on native and cross-linked human hemoglobin as a function of the structure of the lipopolysaccharide A comparison of the effects of smooth and rough lipopolysaccharide Douglas L. Currell and Jack Levin Department of Laboratory Medicine, University of California School of Medicine and Veterans Administration Medical Center, San Francisco, CA, USA The binding of lipopolysaccharide (LPS, also known as bacterial endotoxin) to human hemoglobin is known to result in oxidation of hemoglobin to methemoglobin and hemichrome. We have investigated the effects of the LPSs from smooth and rough Escherichia coli and Salmonella minnesota on the rate of oxidation of native oxyhemoglobin A 0 and hemoglobin cross-linked between the a-99 lysines. For cross-linked hemoglobin, both smooth LPSs produced a rate of oxidation faster than the corresponding rough LPSs, indicating the importance of the binding of LPS to the hemoglobin. The effect of the LPS appeared to be largely on the initial fast phase of the oxidation reaction, suggest- ing modification of the heme pocket of the a chains. For hemoglobin A 0, the rates of oxidation produced by rough and smooth LPSs were very similar, suggestingthe possibility that the effect of the LPSs was to cause dissociation of hemoglobin into dimers. The participation of cupric ion in the oxidation process was demonstrated in most cases. In contrast, the rate of oxidation of cross-linked hemoglobin by the LPSs of both the rough and smooth E. coli was not affected by the presence of chelators, suggesting that cupric ion had previously bound to these LPSs. Overall, these data suggest that the physiological effectiveness of hemoglobin solutions now being developed for clinical use may be decreased by the presence of lipopolysaccharide in the circulation of recipients. Keywords: bacterial endotoxin (lipopolysaccharide); human hemoglobin; oxidation of hemoglobin; cross-linked hemo- globin. The interaction between bacterial lipopolysaccharide (LPS, also known as bacterial endotoxin) and human hemoglobin (Hb) has been shown in previous studies to affect the properties of both the Hb molecule and the LPS [1–3]. The binding of Hb to the smooth LPSs, Escherichia coli 026:B6 and Proteus mirabilis S 1959, was demonstrated and shown to cause disaggregation and an increase of the biological activity of the LPS [1]. In a related study, Hb similarly enhanced activation of Limulus amebocyte lysate and stimulation of endothelial cell tissue factor production by smooth or rough P. mirabilis [2]. Rough LPS lacks the polysaccharide side-chain that is present in the complete (smooth) LPS molecule. In contrast, Limulus amebocyte lysate activation either by lipid A (which consists of a phosphorylated disaccharide backbone with several long- chain fatty acids) or partially deacylated Salmonella minne- sota 595 (Re) LPS was not enhanced in the presence of Hb. The effect of Hb on the LPS and purified lipid A of rough E. coli has been recently investigated, and significant physical changes in the purified lipid A and in the lipid A moiety of intact LPS were reported [4]. The binding of LPS to oxyHb results in the oxidation of theHbtometHbandhemichrome[3].Incontrasttothe lack of effect of Hb on the biological activity of partially deacylated LPS from S. minnesota 595, this LPS was more effective in producing oxidation of Hb than the LPS of either rough S. minnesota 595 or smooth P. mirabilis [3]. To further clarify these structure–function relationships, we have extended these studies to compare the effects of smooth and rough LPSs of E. coli and S. minnesota on the oxidation of native and cross-linked Hb. Because the auto- oxidationofHbhasbeenshowntodependonthepHand the presence of heavy metal cations [5–8], we have also investigated the effects of pH, EDTA and neocuproine on the LPS-mediated oxidation of Hb. MATERIALS AND METHODS Bacterial lipopolysaccharides Smooth E. coli lipopolysaccharide 026:B6 (Westphal method [9]) was obtained from Difco Laboratories (Detroit, MI, USA). Rough E. coli J5 (Rc) and smooth S. minnesota (Galanos method [10]) were generously provided by K. Meyers (RIBI Immunochem Research, Inc., Hamilton, MT, USA). Deep rough S. minnesota 595 (Re) lipopoly- saccharide (Westphal method [9]) was obtained from List Biological Laboratories, Inc. (Campbell, CA, USA). The lipopolysaccharides (5.0–5.9 mg) were suspended in l.0 mL NaCl/P i (0.9% NaCl), pH 7.4, by treatment for Correspondence to J. Levin, V. A. Medical Center (111-H2) 4150 Clement Street, San Francisco, CA 94121, USA. Fax: + 1 415 831 2506, Tel.: + 1 415 750 6913, E-mail: levinj@medicine.ucsf.edu Abbreviations: LPS, lipopolysaccharide; Hb, human hemoglobin. (Received 19 April 2002, revised 11 July 2002, accepted 1 August 2002) Eur. J. Biochem. 269, 4635–4640 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03163.x 5 min in an ultrasonic bath (Branson Ultrasonic Cleaner, Shelton, CT, USA), after initial suspension with a vortex mixer. The LPS suspensions were stored at 0–4 °Cand immediately before use were retreated with the vortex mixer. Reagents LPS-free NaCl/P i was obtained from Irvine Scientific (Santa Ana, CA, USA) and was diluted with deionized water to produce a buffer, pH 7.4, 0.1 M phosphate and 0.15 M NaCl. All other phosphate buffers used were prepared from monobasic NaH 2 PO 4 (Fisher Scientific Co., Fairlawn, NJ, USA) and dibasic K 2 HPO 4 (J. T. Baker Chemical Co., Phillipsburg, NJ, USA) and used as 0.2 M solutions. Tricine was obtained from Sigma Chemical Co. (St Louis, MO, USA)andusedasa0.15 M solution. Neocuproine and EDTA were obtained from Sigma Chemical Co. and used as a 0.01 M aqueous solution and a 0.1 M solution in NaCl/ P i , respectively. Hemoglobin Hemoglobin A 0 ,58mgÆmL )1 , in Ringer’s lactate, pH 8.0, which had been purified by ion-exchange HPLC as described previously [11], was provided by the Blood Research Detachment, Walter Reed Army Institute of Research, Washington, D.C., USA and stored at )70 °C until use. The initial metHb concentration of Hb A 0 was always < 5%. Human Hb, cross-linked between the Lys99 residues of the a chains by treatment of deoxyHb with bis(3,5-dibromosalicyl) fumarate, also was provided by the Blood Research Detachment [12]. The stock solution was 71 mgÆmL )1 in Ringer’s acetate, pH 7.4. It was sterile and essentially LPS-free (< 100 pgÆmL )1 as assessed by the Limulus amebocyte lysate assay [13]) and stored at )70 °C until use. The initial metHb concentration of the cross- linked Hb was always < 7%. Copper analysis All reagents, buffers, Hb stock solutions and LPS suspen- sions (containing 5.0–5.9 mgÆmL )1 LPS) were analyzed for cupric ion by M. Qian in the laboratory of J. W. Eaton, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA, by the method of Makino [14]. The results are presented in Table 1. Oxidation experiments To 360 lL of buffer was added 6.0 lL of a cross-linked Hb solution, 71 mgÆmL )1 ,or7.0lLofaHbA 0 solution, 58 mgÆmL )1 ,andthen80lL of a suspension of LPS, 5.0–5.9 mgÆmL )1 , to produce an LPS/Hb suspension of approximately equal concentrations (mgÆmL )1 ): the final Hb concentration was 0.8–1.0 mgÆmL )1 .Insomeexperi- ments, 4.4 lLEDTA,0.1 M ,or4.4 lL neocuproine, 0.01 M , was added. The absorption spectrum from 400 nm to 800 nm was measured at selected time intervals during a 2-h period, using a Beckman DU-7400 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA, USA). All experiments were carried out at 37 °C. The temperature was maintained by a circulation water bath, Lauda K-2/RD9 (Brinkman Instruments, Westbury, NY, USA). The neces- sary correction for light scattering in all suspensions that contained lipopolysaccharide was performed with a pro- gram in the spectrophotometer software. The relative concentrations of oxyHb, metHb and hemichromes were obtained by the method of Winterbourn [15] from simul- taneous measurements of absorbances at 560, 577 and 630 nm. The major oxidation product was metHb. The amount of hemichrome produced during a 2-h reaction was typically less than 10% (data not shown). The decrease in concentration of oxyHb with time was utilized as a measure of the rate of oxidation of oxyHb. RESULTS The effect of pH on the auto-oxidation of cross-linked Hb was studied. The rate of decrease of the concentration of oxyHb increased as the pH was lowered over the range from pH 9.0–5.8 (data not shown). As was observed previously by others [5], the reaction is biphasic at pH 7.4 and below, with an initial fast phase followed by a slower phase. To determine the optimum pH at which to study the effect of LPS on the oxidation rate, a comparison of the effects of the LPSs of smooth E. coli and rough S. minnesota on the oxidation rate over the pH range 5.8–9.0 was undertaken (data not shown). Because at pH 7.0 both LPSs produced marked but distinguishable effects, all further experiments were carried out at this pH. The contribution of the polysaccharide component of LPS to its effect on the oxidation of cross-linked Hb was then investigated by a comparison of the effects of rough andsmoothLPSsofE. coli, both in the presence and absence of EDTA (Fig. 1). The rate of oxidation was increased in the presence of the LPSs of both the smooth and rough E. coli, but the rate of oxidation in the presence of the LPS of smooth E. coli was much faster than for the LPS from rough E. coli. Although EDTA markedly decreased the rate of auto-oxidation, its effect on the oxidation rate of cross-linked Hb in the presence of LPS was negligible for both the smooth and rough LPSs (Fig. 1). The effect of the LPSs of smooth and rough S. minnesota on the oxidation rate of cross-linked Hb also was compared (Fig. 2). The rate of oxidation in the presence of the LPS of smooth S. minnesota was much faster than in the presence Table 1. Copper concentration in Hb stock solutions, buffers and LPS suspensions. Sample Cu concentration (l M ) Hemoglobin a,a-Hb (71 mgÆmL )1 ) 1.0 Hb A 0 (58 mgÆmL )1 ) 2.5 LPS (5.0–5.9 mgÆmL )1 ) a S. minnesota (R) 1.8 S. minnesota (S) 3.4 E. coli (R) 5.0 E. coli (S) 6.4 Buffers Phosphate buffers, 0.2 M 0.8 Phosphate, 0.1 M buffered-saline, 0.15 M 0.3 Tricine, 0.15 M 0.0 a Cu concentrations are the mean of two determinations. 4636 D. L. Currell and J. Levin (Eur. J. Biochem. 269) Ó FEBS 2002 of the LPS from rough S. minnesota, both in the presence and absence of EDTA. In contrast to the results with the LPSs of E. coli, EDTA decreased the rate of oxidation. However, the rate of oxidation mediated by the smooth LPS was less affected by the presence of EDTA. The rough S. minnesota LPS increased the initial fast phase of the reaction, but decreased the rate of the slow phase of oxidation in the presence of EDTA. A comparison of rough and smooth LPSs of E. coli and S. minnesota in the presence of EDTA revealed that both in the presence and absence of EDTA, the oxidation of cross- linked Hb was faster in the presence of the smooth LPSs (Figs 1 and 2). In addition, the rate of oxidation mediated by the smooth E. coli LPS was faster than that produced by the smooth S. minnesota LPS. The rate of oxidation in the presence of the rough S. minnesota LPS was slower than that produced by the other three LPSs studied. A comparison of the auto-oxidation of cross-linked Hb with that of Hb A 0 is shown in Fig. 3A. The effect of the presence of EDTA, known to bind heavy metal cations [16], on the oxidation of both Hbs is also presented in Fig. 3A. The rate of auto-oxidation of cross-linked Hb was greater than that of Hb A 0, both in the presence and absence of EDTA, as has been observed previously [17]. In addition, the rates of auto-oxidation of both cross-linked Hb and Hb A 0 were markedly reduced by EDTA, suggesting catalysis of the oxidation by heavy metal cations, as previously observed by Rifkind [8,18]. To determine whether the heavy metal cation was cupric ion as indicated by the results of Rifkind [8], the effect of a chelator specific for cupric ion, neocuproine [19,20], was studied. The results in Fig. 3B,C show that the effects of neocuproine and EDTA on the oxidation rate were identical, confirming that the cupric ion was the heavy metal cation primarily responsible for the catalysis. The concen- trations of cupric ion in the solutions used were determined by chemical analysis (Table 1). Fig. 1. Comparison of the effects of the LPSs of smooth E. coli 026:B6 and rough E. coli J5 (Rc), in the absence and presence of EDTA, on the oxidation of a,a-cross-linked Hb (XL Hb). Hb concentration was 0.8 mgÆmL )1 , in phosphate buffer, 0.2 M ,pH7.0.LPSconcentration was 0.8–1.0 mgÆmL )1 . The mean ± SD of three independent experi- ments is shown. Each experiment was performed with aliquots of a single sample of Hb. Therefore, apparent differences in the starting oxyHb concentrations are the result of an immediate drop in the oxyHb concentration upon addition of the LPS. Fig. 2. Comparison of the effects of the LPSs of rough S. minnesota 595 (Re) and smooth S. minnesota, in the absence and presence of EDTA, on the oxidation of a, a-cross-linked Hb. Themean±SDofthreeinde- pendent experiments is shown. Other conditions as in Fig. 1. Fig. 3. The effect of EDTA or neocuproine on the auto-oxidation of a,a-cross-linked Hb (XL) and Hb A 0 (A 0 ). Hb concentration was 0.8 mgÆmL )1 , in phosphate buffer, 0.2 M ,pH7.0.Themean±SDof three or four independent experiments is shown. Ó FEBS 2002 Oxidative effects of LPS on human hemoglobin (Eur. J. Biochem. 269) 4637 Our studies of the effect of the structure of the LPS on the oxidation reaction were extended to native human Hb A 0 to determine whether the above effects were general or specific to cross-linked Hb. A comparison of the effects of the LPSs of rough and smooth E. coli on the oxidation of Hb A 0 , both in the presence and absence of EDTA, is provided in Fig. 4. The rate of oxidation was increased in the presence of the LPSs of both smooth and rough E. coli,butin contrast to the results obtained for cross-linked Hb, the difference in the oxidation rates mediated by the two LPSs was slight. In both cases EDTA reduced the oxidation rate, in contrast to the results obtained with cross-linked Hb in the presence of LPS (Fig. 1), upon which EDTA had no effect. An interesting characteristic of the reaction mediated by the LPS of rough E. coli was a lag phase of 10 minutes both in the presence and absence of EDTA. The lag phase was followed by a very rapid second phase only in the absence of EDTA. Significantly, this lag phase was not observed during the oxidation of cross-linked Hb produced by the LPS of rough E. coli (Fig. 1). The effect of the LPSs of smooth and rough S. minnesota on the oxidation rate of Hb A 0 was compared (Fig. 5). In contrast to the results observed with cross-linked Hb, the rates of oxidation were identical for the LPSs of smooth and rough S. minnesota, both in the presence and absence of EDTA. For both the smooth and rough LPSs, the effect of EDTA was to reduce the oxidation rate. A comparison with the auto-oxidation rate (data from Fig. 3) revealed that in the presence of EDTA, the increase in the rate of oxidation of Hb A 0 produced by the LPSs of rough and smooth S. minnesota was solely due to a sharp increase in the initial rate (Fig. 5). A comparison of the effects of the rough and smooth LPSs of E. coli and S. minnesota on the oxidation of Hb A 0 in the presence of EDTA revealed that the oxidation of Hb A 0 was somewhat faster in the presence of the smooth LPSs (Figs 4 and 5). The rate of oxidation mediated by the smooth E. coli LPS was slightly faster than that produced by the smooth S. minnesota LPS in the presence of EDTA. The rate of oxidation produced by the rough S. minnesota LPS was slower than for the other three LPSs studied. Indeed, the LPS of rough S. minnesota hadnoeffectonthe oxidation of Hb A 0 . The previously reported increase in the oxidation rate of Hb A 0 in the presence of rough S. minnesota [3] was probably due to the presence of cupric ion, as no EDTA was present. DISCUSSION The effect of pH on the auto-oxidation of Hb A 0 has been the subject of several studies. Mansouri and Winterhalter [5] reported that the oxidation of the a chains of Hb A 0 was 10 times faster than that of the beta chains and that the oxidation of the beta chains was not influenced by pH. The biphasic reaction was shown to consist of a rapid initial reaction followed by a slower second phase over a wide pH range from 5.3 to 8. Tsuruga and Shikama [21] confirmed that the fast phase of oxidation was due to the a chains and the slow phase was due to the bchains. Tsuruga et al. found that the beta chain of the tetramer does not exhibit any proton-catalyzed auto-oxidation [22]. These authors found further that upon dissociation of tetrameric oxyHb A 0 into dimers by dilution, the rate of the fast phase was increased markedly while the rate of the slow phase remained unchanged. The observation that cross-linked Hb oxidizes faster than Hb A 0 (Fig. 3A) is consistent with the results of others who reported that the rate of auto-oxidation is inversely propor- tional to the oxygen affinity of the Hb [17]. Therefore, the demonstration of more marked auto-oxidation of the cross- linked Hb than was observed for Hb A 0 can be attributed to the lower oxygen affinity of the cross-linked derivative. The data in Fig. 2 indicate that at pH 7.0, in the presence of EDTA, the oxidation of cross-linked Hb mediated by the LPS of rough S. minnesota was very slow. In contrast, the effect on the oxidation reaction of the LPS of smooth E. coli was marked and not altered in the presence of EDTA (Fig. 1). The binding of the LPS molecule of the smooth E. coli to Hb, shown previously in this laboratory [1], apparently increases the oxidation rate while shielding the Hb from the effect of heavy metal cations, perhaps through binding of the cations by the LPS. Support for this idea is provided by the report of the binding of 1.5–2 mol of iron in either the ferrous or ferric state to the LPS of smooth E. coli. Such binding Fig. 4. Comparison of the effects of the LPSs of smooth E. coli 026:B6 and rough E. coli J5 (Rc), in the absence and presence of EDTA, on the oxidation of Hb A 0 . The mean ± SD of three independent experi- ments is shown. Other conditions as in Fig. 1. Fig. 5. Comparison of the effects of the LPSs of rough S. minnesota 595 (Re) and smooth S. minnesota, in the absence and presence of EDTA, on the oxidation of Hb A 0 . The mean ± SD of three independent experiments is shown. Other conditions as in Fig. 1. 4638 D. L. Currell and J. Levin (Eur. J. Biochem. 269) Ó FEBS 2002 resulted in a slight decrease in the biological activity of the LPS [23]. The results show that for both S. minnesota and E. coli, the smooth (wild type) LPS was more effective in increasing the oxidation rate of cross-linked Hb than the rough LPSs lacking the O-specific polysaccharide moiety (Fig. 6). It was previously found that the singly deacylated derivative of rough S. minnesota 595 was more effective than the rough LPS [3]. Partial deacylation probably disturbs the supra- molecular structure of the rough LPS, exposing the fatty acids of the lipid A component. Therefore, it was suggested that the lipid A moiety (Fig. 6) is crucial in catalyzing the oxidation of Hb [3]. However, as the lipid A constitutes a much smaller proportion of the molecular mass of smooth LPSs, their greater effect on the oxidation rate may be due to more effective binding of Hb. The effects of the various LPSs on the oxidation rate of cross-linked Hb can be compared in Figs 1 and 2. The relative rates both in the presence and absence of EDTA are: smooth E. coli > smooth S. minnesota > rough E. coli >roughS. minne- sota. In the presence of EDTA, the oxidative effect of the rough E. coli LPS approaches that of the smooth S. min- nesota LPS. The results of experiments, in which the oxidation of Hb A 0 by the four LPSs was studied, indicated that their effects upon Hb A 0 and cross-linked Hb differ (Figs 4 and 5 vs. Figs 1 and 2). This difference in behavior of native human Hb A 0 and cross-linked Hb, in the presence of the LPSs utilized, must lie in the principal differences in the properties of the two Hbs [24]. The reduced oxygen affinity of the cross-linked Hb would be expected to maximize any effects due to the resultant increase in oxidation rate. At equilibrium, the concentration of deoxyHb is much greater in the low affinity cross-linked Hb. Therefore, another difference between cross-linked Hb and Hb A 0 may be in the binding of the deoxyHb to the LPS. Another obvious difference between the two Hbs is the possibility of dissociation of the Hb A 0 into dimers, which is not possible for the cross-linked Hb, as cross-linking the a chains prevents dissociation into ab dimers. Dissociation into dimers is known to increase the oxidation rate of Hb [25]. Thus, if the oxidative effect of LPS on native human Hb A 0 is primarily due to the enhancement of the dissociation of the Hb into dimers, then the observed rates of oxidation caused by each of the LPSs studied should be similar to each other, i.e. simply that of the rate of oxidation of dimers. The increase in rate of oxidation in the presence of rough E. coli was striking for Hb A 0 (Fig. 4). This effect was reduced in the presence of EDTA, suggesting that the binding of heavy metal cations to smooth E. coli may be greaterthantoroughE. coli, as binding of heavy metal cations to the LPS would be expected to prevent catalysis of oxidation by the cations. However, it is not clear why the effect of EDTA was negligible in the oxidation of cross- linked Hb mediated by the rough LPS of E. coli (Fig. 1). The mechanism by which LPSs accelerate the oxidation of cross-linked Hb is not clear. Because the achains are covalently linked, dissociation into ab dimers is not possible. It is conceivable that binding of the Hb tetramer to the LPS molecule makes the heme cavity of the a chains more accessible to a water molecule which can then accelerate the displacement of the protonated superoxide anion, as was suggested by Tsuruga and Shikama [21] to explain the increase in oxidation rate of the a chains in the ab dimer. It had been demonstrated earlier by Wallace et al. [26] that nucleophiles such as water are important in the proton assisted displacement of superoxide during the auto-oxida- tion of Hb. In general, the increase in the oxidation rate of cross- linked Hb mediated by LPSs is due to an increase in the rate of the initial fast phase, i.e. oxidation of the a chains. The rates of oxidation are reduced in the presence of chelators of heavy metal cations in most cases. An exception is the lack of alteration of the increased oxidation rates of cross-linked Hb in the presence of the LPSs of smooth or rough E. coli. This lack of effect of the chelator suggests that the LPS itself binds the heavy metal cations (probably at the phosphate groups) and thus prevents catalysis of Hb oxidation by the heavy metal cations. For cross-linked Hb, the smooth LPSs were more effective than the rough LPSs, suggesting that binding of the Hb by the LPS was more important than the lipid content of the LPS. Overall, the E. coli LPSs were more effective than the S. minnesota LPSs in increasing the rate of oxidation, suggesting a difference in binding of the two types of LPSs. The effect of the LPSs on the rate of oxidation of Hb A 0 was much less than on cross-linked Hb and furthermore, the differences in structures of the LPSs were less important, suggesting that the effect of the LPS on Hb A 0 was possibly due to enhancement of dissociation into dimers. An exception to the above general statements is the behavior of the LPS of rough E. coli. This LPS exhibited a lag phase of approximately 10 min, followed by a very rapid phase of oxidation for Hb A 0 but not for cross- linked Hb. Furthermore, the rate of the oxidation reaction mediated by this LPS was decreased by EDTA for Hb A 0 but not for cross-linked Hb. It is possible that the binding of Hb A 0 by the LPS of rough E. coli exposes the binding site for heavy metal cations on the Hb which then leads to catalysis of oxidation. The b-2 histidine has been sugges- ted as the binding site for cupric ion in Hb [18], but its distance from a heme makes it difficult to understand its involvement in the oxidation process. Another potential binding site, the b-93 sulfydryl, is close to the heme [27] and thus more likely to be involved in the oxidation of Hb. Indeed, interaction between cupric ion bound to the b-93 sulfhydryl and the heme center has recently been demonstrated [28]. Many types of preparations of Hb, including cross-linked Hb, are now under development as red blood cell substitutes [29]. It is likely that LPS will be present in the circulation of many of the potential recipients of Hb solutions, as endotoxemia may occur in patients who are hypotensive Fig. 6. Schematic representation of smooth LPS, rough LPS and lipid A. Ó FEBS 2002 Oxidative effects of LPS on human hemoglobin (Eur. J. Biochem. 269) 4639 and/or have experienced trauma and hemorrhage. The studies described in this investigation, in conjunction with our previous reports of the effects of LPS on both native and cross-linked Hb [1,3], suggest the possibility that the presence of circulating LPS may significantly decrease the ability of Hb solutions to satisfactorily function as oxygen carriers. ACKNOWLEDGEMENTS Supported in part by the Veterans Administration and the REAC Committee of the University of California School of Medicine, San Francisco. REFERENCES 1. Kaca, W., Roth, R.I. & Levin, J. (1994) Hemoglobin, a newly recognized lipopolysaccharide (LPS)-binding protein that enhances LPS biological activity. J. Biol. Chem. 40, 25078– 25084. 2. Kaca, W., Roth, R.I. & Levin, J. (1994) Human hemoglobin increases the activity of bacterial lipopolysaccharides in activation of Limulus amebocyte lysate and stimulation of tissue factor production by endothelial cells in vitro. J. Endotoxin Res. 1,243– 252. 3. Kaca, W., Roth, R.I., Vandegriff, K.D., Chen, G.C., Kuypers, F.A.,Winslow,R.M.&Levin,J.(1995)Effectsofbacterial endotoxin on human cross-linked and native hemoglobins. Biochemistry 34, 11176–11185. 4. Ju ¨ rgens, G., Mu ¨ ller, M., Koch, M.H.J. & Brandenburg, K. (2001) Interaction of hemoglobin with enterobacterial lipopolysaccharide and lipid A. Physicochemical characterization and biological activity. Eur. J. Biochem. 268, 4233–4242. 5. Mansouri, A. & Winterhalter, K.H. (1973) Nonequivalence of chains in hemoglobin oxidation. Biochemistry 12, 4946–4949. 6. Brooks, J. (1931) The oxidation of haemoglobin to methaemo- globin by oxygen. Proc. Roy. Soc., Series B 109, 35–50. 7. Brooks, J. (1935) The oxidation of haemoglobin to methaemo- globin by oxygen. II. The relation between the rate of oxidation and the partial pressure of oxygen. Proc. Roy. Soc., Series B 118, 560–577. 8. Rifkind, J.M. (1974) Copper and autoxidation of hemoglobin. Biochemistry 13, 2474–2481. 9. Westphal, O. & Jann, K. (1965) Bacterial lipopolysaccharides. Extraction with phenol-water and further application of the pro- cedure. Methods Carbohydrate Res. 5, 91–93. 10. Galanos, C., Luderitz, O. & Westphal, O. (1969) A new method for the extraction of R-lipopolysaccharides. Eur. J. Biochem. 9, 245–249. 11. Winslow, R.M. & Chapman, K.W. (1994) Pilot-scale preparation of hemoglobin solutions. Methods Enzymol. 231, 3–16. 12. Christensen, S.M., Medina, R., Winslow, R.M., Snell, S.M., Zegna, A. & Marini, M.A. (1988) Preparation of human hemoglobin A 0 for possible use as a blood substitute. J. Biochem. Biophys. Methods 17, 145–154. 13. Levin, J. & Bang, F.B. (1968) Clottable protein in Limulus: its localization and kinetics of its coagulation by endotoxin. Thromb. Diath. Haemorrh. 19, 186–197. 14. Makino, T. (1989) A sensitive direct colorimetric assay of serum copper using 5-Br-PSAA. Clinica Chimica Acta 185, 7–16. 15. Winterbourn, C.C. (1990) Oxidative reactions of hemoglobin. Methods Enzymol. 186, 256–274. 16. Sille ´ n, L.G. & Martell, A.E. (1964) Stability Constants of Metal- Ion Complexes, pp. 625–626. The Chemical Society, London. 17. MacDonald, V. (1994) Measuring relative rates of hemoglobin oxidation and denaturation. Methods Enzymol. 231, 480–490. 18.Rifkind,J.M.,Lauer,L.D.,Chiang,S.C.&Li,N.C.(1976) Copper and the oxidation of hemoglobin: a comparison of horse and human hemoglobins. Biochemistry 15, 5337–5343. 19. Nebesar, B. (1964) Spectrophotometric determination of copper in tellurium and related thermoelectric compounds of the bismuth telluride type with 2,9-dimethyl-1,10-phenanthroline. Anal. Chem. 36, 1961–1965. 20. Stephens, B.G., Felkel, H.L. & Spinelli, W.M. (1974) Spectro- photometric determination of copper and iron subsequent to the simultaneous extraction of bis (2,9-dimethyl-1,10-phenanthroline) copper (l) and bis[2,4,6-tripyridyl)-1,3,5-triazine]iron (ll) into propylene carbonate. Anal. Chem. 46, 692–696. 21. Tsuruga, M. & Shikama, J. (1997) Biphasic nature in the auto- xidation reaction of human oxyhemoglobin. Biochim. Biophys. Acta 1337, 96–104. 22. Tsuruga, M., Matsuoka, A., Hachimori, A., Sugawara, Y. & Shikama, K. (1998) The molecular mechanism of autoxidation for human oxyhemoglobin. J. Biol. Chem. 273, 8607–8615. 23. Roth, R.I., Panter, S.S., Zegna, A.I., Arellano, F.A. & Levin, J. (1997) Effects of iron on bacterial endotoxin. J. Endotoxin Res. 4, 273–278. 24. Chatterjee, R., Welty, V., Walder, R.Y., Pruitt, S.L., Rogers, P.H., Arnone, A. & Walder, J.A. (1986) Isolation and characterization of a new hemoglobin derivative cross-linked between the a chains (lysine 99a 1 fi lysine a 2 ). J. Biol. Chem. 261, 9929–9937. 25. Zhang, L., Levy, A. & Rifkind, J.M. (1991) Autoxidation of hemoglobin enhanced by dissociation into dimers. J. Biol. Chem. 266, 24698–24701. 26. Wallace, W.J., Maxwell, J.C. & Caughey, W.S. (1974) The mechanisms of hemoglobin autoxidation: evidence for proton- assisted nucleophilic displacement of superoxide by anions. Biochem. Biophys. Res. Comm. 57, 1104–1110. 27. Muirhead, H., Cox, J.M., Mazzarella, L. & Perutz, M.F. (1967) Structure and function of haemoglobin III. A three-dimensional Fourier synthesis of human deoxyhaemoglobin at 5.5 A ˚ resolu- tion. J. Mol. Biol. 28, 117–156. 28. Bonaventura, C., Godette, G., Tesh, S., Holm, D.E., Bonaventura, J., Crumbliss, A.L., Pearce, L.L. & Peterson, J. (1999) Internal electron transfer between hemes and Cu (II) bound at cysteine b93 promotes methemoglobin reduction by carbon monoxide. J. Biol. Chem. 274, 5499–5507. 29. Stowell, C.P., Levin, J., Spiess, B.D. & Winslow, R.M. (2001) Progress in the development of RBC substitutes. Transfusion 41, 287–299. 4640 D. L. Currell and J. Levin (Eur. J. Biochem. 269) Ó FEBS 2002 . The oxidative effect of bacterial lipopolysaccharide on native and cross-linked human hemoglobin as a function of the structure of the lipopolysaccharide A. compare the effects of smooth and rough LPSs of E. coli and S. minnesota on the oxidation of native and cross-linked Hb. Because the auto- oxidationofHbhasbeenshowntodependonthepHand the