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Differential Prooxidative Effect of Adult and Fetal Hemoglobin

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Differential Prooxidative Effect of Adult and Fetal Hemoglobin View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 196, number 2 FEES 3395 February 1986 Differential prooxidative effect of adult and fetal hemoglobin Alfonso Valenzuela, Ricardo Guerra, Laura Lazcano and Patricia Caiias Laborutorio de Bioquimica, Institute de Nutrici6n y Tecnologia de 10s Alimentos, Universidad de Chile, Casilla 15138, Santiago 1 I, Chile Received 13 December 1985 Human fetal hemoglobin assayed in a peroxidizing system shows an increased prooxidative effect when com- pared to human adult hemoglobin. This effect is related only to the oxyhemoglobin form since both fetal and adult methemoglobins did not show any prooxidative effect. The prooxidative effect of the oxyhemo- globin form is ascribed to its increased sensitivity to form superoxide free radicals when transformed to the methemoglobin form. It is proposed that the structure of the heme pocket of fetal oxyhemoglobin en- hances free radical release when compared to adult oxyhemoglobin, This difference may be important in some hematological disorders presented by the newborn. Oxyhemoglobitz Lipid peroridation Methemoglobin Hemoglobin 1. INTRODUCTION hemoglobin in its oxy- and methemoglobin states using a peroxidizing system formed by potassium Structural differences between adult and fetal linoleate. hemoglobin are determined by the presence of ar-fi chains in adult hemoglobin (HbA) and (~-7 chains 2. EXPERIMENTAL in fetal hemoglobin (HbF). The oxygen affinity of both hemoglobins is very different, HbF having an Samples were obtained from the cord blood of increased saturation capacity compared to HbA at 10 full-term newborn infants and from the venous low oxygen tensions. blood of their mothers (Hospital Paula Jara- Human fetal erythrocytes are more susceptible Quemada, Santiago). All newborn were normal to oxidative hemolysis than adult erythrocytes [I]. and their mothers presented no evidence of hema- The antioxygenic capacity of fetal erythrocytes is tological diseases. Blood samples were collected in different from that of adult red cells. Comparative heparinized tubes, centrifuged at 1500 x g for studies of maternal and cord blood show that the 10 min and the plasma and buffy coat removed by activity of the enzymes catalase and glutathione aspiration. Red cells were washed twice with saline peroxidase is significantly lower in fetal erythro- and lysed by the addition of 1.5 vols distilled cytes while that of the enzyme superoxide dismutase water. Cell debris was discarded by centrifugation is equivalent to that found in maternal erythro- at 6500 x g for 15 min and the hemoglobin super- cytes [Z]. In addition, HbF exhibits an increased natant of each sample was fractionated by chro- sensitivity relative to HbA to form methemoglobin matography on CM-Sepharose to separate HbA 131 through the univalent reduction of oxygen and HbF. Fractionation was according to Congote leading to the formation of the prooxidant super- and Kendall [5] based on the different elution pat- oxide free radical [4]. tern of both hemoglobins to an NaCl concentra- This study was undertaken to investigate the tion gradient. After elution, fractions containing prooxidant properties of adult and fetal human each hemoglobin type were collected, dialyzed Published by Elsevier Science Publishers B. K (Biomedical Division) 00145793/86/$3.50 0 1986 Federation of European Biochemical Societies 353 Volume 196, number 2 FEBS LETTERS February 1986 against distilled water and concentrated by ultra- carded and the major fractions converted, after filtration using a dialysis tube (Spectrator mem- collection, to the oxy- and methemoglobin forms. brane tubing 0%667A) under negative pressure at Neither fractions presented any detectable free 5°C to l/IO of its origina volume. After concen- Fe2” and were absent of superoxide dismutase and tration each fraction was suspended in phosphate- catalase activities. buffered saline (9 parts 0.9% NaCl, 1 part 0.1 mM KHzPO~, 50mM EDTA; pH 7.4) to a final con- 3.2. comparative effect of HbF and HbA on the centration of 10mg Hb/ml. Hemoglobin concen- oxyhemoglobin and methemoglobin forms tration was determined as cyanmethemoglobin [6]. in the Iinoieate peroxidation system Oxyhemoglobin was prepared by bubbling 0~ Fig.2 shows the prooxidant effect of HbF and (100%). Methemoglobin was formed by incubating HbA on their oxy forms in the linoleate peroxi- a soIution of hemoglobin with an equal volume of dizing system. Both hemoglobins elicited a concen- 1% NaNOz in the phosphate-buffered saline for tration-dependent increase in linoleate peroxida- 10min at room temperature. Excess nitrite was tion vs controls, HbF showing a signi~cantIy removed by passing the methemoglobin solution higher prooxidant effect than HbA. On the other through a Sephadex G-25 column in phosphate- hand, the results obtained with the methemoglobin buffered saline. Superoxide dismutase and catalase forms of both fractions show that HbF and HbA activity of the hemoglobin solutions was assayed elicited only a slight prooxidant effect, this being according to Winterbourn et al. [7] and Beers and of a similar magnitude at the same hemoglobin Sizer [S], respectively. Free Fe’+ in the hemoglobin concentrations (table 1). This effect is in accor- solutions was determined using a diagnostic Merck dance with Szebeni et al. [12] who described the test (Merckotest no. 3307). The potassium linoleate absence of pero~dation in hemosomes containing peroxidizing system was prepared as in [9] using a methemoglobin and formed by unsaturated lipids. peroxide-free linoleic acid and the peroxidation The higher prooxidative effect of oxy HbF may initiated by adding the oxy- and methemoglobin be the result of its increased sensitivity to be con- solutions. Linoleate peroxidation was assessed by verted to methemoglobin with the release of hydro- conjugated diene formation at 37% and measured gen peroxide and superoxide free radicals [ 131. at 233 nm according to Hasse and Dunkley [IO]. Re- These radicals and the related hydroxyl free radical sults were expressed as nmol conjugated dieneslml are involved in the initiation and propagation of peroxidizing system using t 2.52 x 1091*mol-’ unsaturated fatty acid peroxidation 1141. Linoleate [li]. All reagents were obtained from Sigma (St. peroxidation can be inhibited by superoxide dis- Louis, MO). Results are expressed as means -t SE and the significance of the differences between 06 - MATERNAL BLOOD -0 16 ---- CORD BLOOD E mean values was assessed by Student’s t-test for c unpaired data. 3. RESULTS AND DISCUSSION 3.1. Separation of HbF and HbA from maternal and fetal cord blood hemolysates The separation of HbF and HbA from fetal to adult blood on CM-Sepharose is shown in fig.1. Both hemoglobins can be fractionated when an NaCl concentration gradient is appIied to the col- 20 40 60 80 100 umn. The absorbance profile indicates the. pres- 0 FRACTION NUMBER 15ml 1 ence of two major fractions in both hemolysates, corresponding to the HbF and HbA components Fig.1. Separation of HbF and HbA from maternal as described by Congote and Kendall [5]. The and cord blood hemolysates by chromatography on minor components of the HbF and HbA present in CM-Sepharose. Elution profile represents a typical adult and fetal hemolysates respectively were dis- fractionation pattern. 354 Volume 196, number 2 FEBS LETTERS February 1986 tions on the heme pocket of oxyhemoglobin may allow access of water molecules to the heme, re- sulting in the formation of methemoglobin and release of superoxide radicals. Any factor capable of causing increased flexibility or distortion of the heme pocket could result in increased methemoglo- bin formation. This situation has been observed with redox dyes (e.g. ascorbate, phenylhydrazine) and in some unstable hemoglobins [ 171. We are aware of no report describing such an effect for fetal hemoglobin. Our results on the prooxidant effect of HbF sustain the view that the structure of the heme pocket of fetal hemoglobin may result in an enhanced effect on its methemoglobin conver- sion when compared to adult hemoglobin. This effect may be an important component in some 0 IO 20 30 40 hematological disorders observed in the newborn, TIME (min 1 especially in cases of premature delivery, where the cellular defenses against oxygen toxicity are not Fig.2. Comparative effect of different concentrations of fully developed [2]. HbF and HbA in the oxyhemoglobin form on linoleate peroxidation. Hemoglobin concentration is expressed as pg/ml peroxidizing system. Control values correspond to ACKNOWLEDGEMENTS linoleate peroxidation obtained without oxyhemoglobin addition. This research was supported by grants B-1765 8533 and B-2019-8522 from Direction de Investi- gacion y Bibliotecas, Universidad de Chile. We are Table 1 grateful to Cecilia Gomez who typed the manu- Effect of the methemoglobin form of fetal hemoglo- script. bin (HbF) and adult hemoglobin (HbA) on linoleate peroxidation after 40min incubation at 37’C REFERENCES Addition/ml nmol conjugated dienes/ml 111Oski, F.A. and Berness, LA. (1967) J. Pediatr. 79, peroxidizing system peroxidizing system 21 l-220. 121Agostoni. A., Gerli, G.L., Beretta, L. and Bianchi, HbF (1Opg) 6.2 + 0.18 M. (1980) J. Clin. Chem. Clin. Biochem 18, 771- HbA (10 /g) 6.8 + 0.22 773. HbF (5O~g) 7.1 f 0.14 131Wind, M. and Stern, A. (1977) Experientia 33, HbA (50 cg) 6.9 f 0.08 1500-1501. Control 5.9 * 0.42 (41 Misra, H.P. and Fridowich, I. (1972) J. Biol. Chem. 247, 6960-6962. Results represent the average of 6 experiments + SE. VI Congote, L.F. and Kendall, G. (1982) Anal. Bio- Control values correspond to linoleate peroxidation them. 123, 124-132. obtained without methemoglobin addition. Differences b51 Drabkin, D.L. and Austin, J.M. (1939) J. Biol. between each value of HbF or HbA and the control are Chem. 112, 52-56. not significant 171 Winterbourn, C., Hawkins, R., Brian, M. and Carrel, W.
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