- Email: [email protected]
Spectroscopic study of gamma irradiated bovine hemoglobin
ARTICLE IN PRESS
Radiation Physics and Chemistry 76 (2007) 1600–1605 www.elsevier.com/locate/radphyschem
Spectroscopic study of gamma irradiated bovine hemoglobin Ahmed Mohamed Maghrabya,, Maha Anwar Alib a
Radiation Dosimetry Department, National Institute of Standards (NIS), NIS-Tersa Street 12211, Haram-Giza, P.O. Box 136, Egypt b Biophysics Department, Faculty of Science, Cairo University, Giza, P.O. Box 12613, Egypt Received 14 November 2006; accepted 21 January 2007
Abstract In the present study, the effects of ionizing radiation of Cs-137 and Co-60 from 4.95 to 743.14 Gy and from 40 Gy to 300 kGy, respectively, on some bovine hemoglobin characteristics were studied. Such an effect was evaluated using electron paramagnetic resonance (EPR) spectroscopy, and infra-red (IR) spectroscopy. Bovine hemoglobin EPR spectra were recorded and analyzed before and after irradiation and changes were explained in detail. IR spectra of unirradiated and irradiated Bovine hemoglobin were recorded and analyzed also. It was found that ionizing radiation may lead to the increase of free radicals production, the decrease in a-helices contents, which reflects the degradation of hemoglobin molecular structure, or at least its incomplete performance. Results also show that the combined application of EPR and FTIR spectroscopy is a powerful tool for determining structural modification of bovine hemoglobin samples exposed to gamma irradiation. r 2007 Elsevier Ltd. All rights reserved. Keywords: EPR; ESR; Radiation; Bovine; Hemoglobin
1. Introduction The biological effects of radiation could be analyzed in part by the study of the primary processes of radiationinduced damage of vitally important proteins. Hemoglobin (Hb) is the important iron-containing oxygen transport protein in the body; therefore, radiation-induced damage of Hb is a subject of various studies (Teicher et al., 1998; Geoffrey and Jacobs, 1998; Dreval’ and Sichevskaia, 2000; Puchala et al., 2004; Montebello et al., 2005). The study of the effect of ionizing radiation on the molecular structure of Hb is quite important for several reasons; one of them is to investigate the effect of probable changes in Hb molecules after radiation therapy sessions or even in cases of diagnostic applications. It is important also for occupational radiation workers and in emergency or accident cases, where the use of complete blood count (CBC) does not indicate the functionality of Hb content while it may possess normal count (Farley et al., 1995). Also, the study of some properties of heme may be an Corresponding author. Tel.: +2 0124509723; fax: +2 023867451.
E-mail address: [email protected] (A.M. Maghraby). 0969-806X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2007.01.008
useful indicator for its denaturation (Wajnberg and Bemski, 1993). For erythrocyte proteins, Hb normally account for about 90% and spectrin for about 10%. Comporti et al. (2002) showed that Hb denaturation occurred after irradiation and this protein is the most susceptible to reactive oxygen species attacks. Although it was studied before (Pelletier et al., 1978), the action of ionizing radiation on Hb was not reported quantitatively (at the knowledge of authors), and the limit and the kind of the probable effect(s) were not provided accurately, hence there is a need to determine such effect(s) quantitatively and accurately. Therefore, the aim of the present in vitro study was to study the effect of ionizing radiation on Hb molecule itself and eliminating other contributing factors that may lead to the enhancement or suppression of this effect. Spectrophotometric properties of bovine Hb are only slightly different from those of human blood (Zijlstra and Buursma, 1997). Its importance may be reflected by the interest in the development of safe, and effective substitute for human erythrocytes as a transfusible medium for oxygen transport. Polymerized forms of bovine Hb, such as
ARTICLE IN PRESS A.M. Maghraby, M.A. Ali / Radiation Physics and Chemistry 76 (2007) 1600–1605
1601
HBOC-201, show particular promise. They have a molecular structure similar to that of human Hb but have lower concentrations of organic phosphates, resulting in more pronounced oxygen unloading in ischemic tissue and increased Hb binding of carbon dioxide in the deoxygenated state (Mullon et al., 2000). Moreover, some foods (especially children foods) may contain bovine Hb concentrate, designed to provide certain quantity of bio-available iron per day (Zijlstra and Buursma, 1997), hence this food (or its components) may require doses of about 30–50 kGy for industrial sterilization. Therefore, in the present study the effect of ionizing radiation of Cs-137 and Co-60 from 4.95 to 743.14 Gy (clinical usages) and from 40 Gy to 300 kGy (industrial use), respectively, on some bovine Hb characteristics was studied. 2. Materials and methods 2.1. Irradiation facilities Cesium-137 gamma source (model: GB-150-Canada), was used for samples irradiation. It was manufactured by Atomic Energy of Canada Limited on April 1970 with original activity of 1000 Ci (3.7 1010 kBq), dose rate were about 1.2 Gy/h at 1 m. Although the half-life time of the source is not short (30.17 a), doses were corrected for source decay each irradiation turn. Irradiation was also performed using a Co-60 g-source (irradiation cell) installed at the National Center for Radiation Research and Technology (NCRRT) at the Egyptian Atomic Energy Authority. It possesses activity of 50,000 Ci, with dose rate of about 4.0 kGy/h. Samples were irradiated at room temperature in a Perspex phantom irradiation capsules.
Fig. 1. Bovine Hb is composed of two pairs of non-identical subunits, alpha and beta. Each alpha–beta pair is more closely associated than they are with each other, but the overall arrangement is roughly tetrahedral.
methemoglobin (http://www.sigmaaldrich.com/catalog/search/ ProductDetail/FLUKA/51292). Structure of bovine Hb is shown in Fig. 1. It is composed of two pairs of non-identical subunits, alpha and beta. Each alpha–beta pair is more closely associated than they are with each other, but the overall arrangement is roughly tetrahedral (http://www.bmb.uga.edu/ wampler/tutorial/prot4.html) (Marta et al., 1996). Powder Hb samples were pressed into disks and then were wrapped in thin-sealed plastic bags prior to irradiation in order to minimize the possibility of interaction between samples and molecular oxygen. 2.4. Evaluation method of EPR spectra
2.2. Radiation dose determination Air kerma (Kair) was determined according to the International Atomic Energy Agency (IAEA) code of practice TRS-(381) (IAEA, 1997). Kair determination was performed using the secondary standard dosimetry system of the National Institute of Standards (NIS), Egypt, which is composed of a NPL-NE2560 electrometer (UK) and a NE2561 ionization chamber (UK). The secondary standard calibration system was calibrated at the Bureau International des Poids et Mesure (BIPM). The expanded uncertainty associated to Kair determination was about 0.9% at 95% level of confidence (coverage factor ¼ 2). 2.3. Bovine Hb samples Powder bovine Hb of average molecular weight about 64,500 was obtained from Sigma (St. Louis, MO, USA) and used without further purification. Since native Hb is readily oxidized in air, this preparation may be predominantly
The EPR system used in this study is an EMXBRUKER system, Germany, supplied by a 9.5 GHz microwave (X-band) Gunn-Oscillator Bridge with automatic tuning capability and a rectangular 4102 ST cavity operating in the TE102 mode. The standard EMX Signal Channel can be operated at any modulation frequency between 6 and 100 kHz, and has unsurpassed phase resolution and stability. EPR spectra of the empty tubes were recorded before recording the samples spectra in order to ensure the purity of the obtained signals. Readings were corrected to the peak-to-peak height of the reference standard material (Pyrolyzed sucrose) in order to correct for the change in the spectrometer sensitivity. Sample weight was about 72.00712 mg and sample mass normalization was performed for each acquired signal intensity. The spectrum of each single sample was recorded three or more successive times. All measurements were performed at room temperature.
ARTICLE IN PRESS A.M. Maghraby, M.A. Ali / Radiation Physics and Chemistry 76 (2007) 1600–1605
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Parameters used for spectra acquisition are as follows: modulation amplitude is 2 mT, modulation frequency is 100 kHz, microwave power is 1.586 mW, center field is 348 mT, sweep width is 20 mT, time constant is 81.92, and conversion time is 40.96 ms for 1024 data points resulting in sweep time about 42 s. 2.5. FTIR spectra acquisition FTIR spectra were recorded using a double-beam IR spectrometer of type Jasco-460 FTIR plus, Japan. Each spectrum is recorded over wavenumber range 4000– 400 cm1. Hb sample is mixed intimately with finely powdered KBr and the mixture is squeezed in a press to about 1000 atm. Under these conditions, the KBr becomes glassy and forms a thin translucent disk in which the finely ground sample is suspended. 3. Results and discussion 3.1. Bovine Hb EPR spectral features Fig. 2 represents the EPR spectra of unirradiated (solid line) and 743 Gy gamma irradiated sample (dotted line). Major features of Bovine Hb EPR spectrum are comparable to the human one (Ikeya, 1993). About four features comprise the spectrum of g-factor equal to 5.910170.0860, 4.275070.0031, 2.147370.0227 and 2.005570.0010, respectively. The first two signals are due to Fe (III) of high spin form (S ¼ 5/2). The first signal (S1) is associated with oxidized heme iron, which clarifies its indication to methemoglobin (MetHb), in which a water molecule replaces O2 ion as a ligand of iron (Wajnberg and Bemski 1993). The second signal (S2) is corresponding to non-heme Fe(III) ions at sites endowed with rhombic symmetry, which is not associated with species involved in blood, such as transferrin which causes an EPR signal near g ¼ 4.3
(S´lawska-Waniewska et al., 2004). From Fig. 2, it is clear that S2 is greater than S1, while this is not true in case of human Hb spectrum (Ikeya, 1993). This means higher nonheme iron content in bovine Hb than that of human. The nature of bovine Hb itself or the deterioration of its molecular structure and hence the decomposition of heme during sample preparation may give reason for the increase of non-heme iron content. Third signal group (S3) is associated with low spin derivatives of ferrihemoglobin called ‘‘hemichrome’’, copper proteins and some transition-metal complexes (Rachmilewitz et al., 1971). Hemichromes are low spin derivatives of ferric Hb brought about through discrete reversible or irreversible changes of protein conformation (Venkatesh et al., 1997). The purity of our sample is about 95%, hemichromes are not easily to be separated from Hb molecules but the rest which is about 5% may contain some of these hemichromes that produce S3. Changes in normal Hb under the effect of time, pH and protein denaturants such as urea or salicylate can form different kinds of hemichromes with different endogenous ligands; hence hemichromes form the primary step to the destructive pathway for denaturation (Venkatesh et al., 1997). With regard to the fourth signal (S4), it appears as a singlet with no hyperfine structures as shown in Fig. 2. This signal is attributed to free radicals in hemoglobin formed by the degradation of blood constituents (Miki et al., 1987). Several investigators agree that at least two different kinds of radicals are formed on the protein (Kelman et al., 1994; Gunther et al., 1995). Although the formation of peroxyl radicals is well proven (Ikeya, 1993; Miki et al., 1987), this kind of radical constitutes only a fraction of the total concentration of radicals (Svistunenko et al., 1997a, b). The globin-based free radical (HB(Fe(IV)QO)) was suggested to be major contributor for S4 (Svistunenko et al., 1997a,b). Many investigations revealed that it is the
Fig. 2. EPR spectra of unirradiated and 743 Gy gamma irradiated bovine Hb samples recorded at room temperature.
ARTICLE IN PRESS A.M. Maghraby, M.A. Ali / Radiation Physics and Chemistry 76 (2007) 1600–1605
Fig. 3. Radiation-induced Tyrosine (Tyr) radical in the bovine Hb (from Svistunenko et al., 2002).
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from the cystein residues appear around 2550 cm1. The bands between 2872 and 3000 cm1 are the symmetric and asymmetric –CH2 and –CH3 stretching vibrations from protein side chains. Other protein bands are the amide B band at about 3050 cm1 and amide A at about 3309 cm1. The amide band most widely used in studies of protein secondary structure is the amide I mode, which has been used extensively to quantify a-helices, b-sheets, turns, and non-ordered structures in proteins. Overlapping components in the amide I band includes ahelix (1657 cm1) or parallel and antiparallel b-sheets (1640 and 1680 cm1, respectively). The vibration of the tyrosine ring (about 1517 cm1) originates from the side chains of the protein overlaps in the amide II band (Gunther et al., 1995). The tyrosine residues adjacent to the C termini of Hb subunits are expected to play important structural roles, because the C termini are the loci of T-state quaternary salt-bridges, and because the tyrosine side chains bridge the H and F helices via H bonds to the carbonyl groups (Kneipp et al., 2006). 3.3. Radiation-induced changes in the bovine Hb EPR spectra
Fig. 4. IR spectra of unirradiated and 743 Gy gamma irradiated bovine Hb samples, recorded at room temperature.
tyrosine (Tyr) radical (shown in Fig. 3) (Svistunenko et al., 1997a,b, 2002, 2004; Svistunenko, 2005) and showed the resemblance between this signal and that of the formed radical after addition of hydrogen peroxide to the met form of Hb: FeIII
R + H2O2
FeIV = OR+ + H2O
(1)
3.2. FTIR spectral features of bovine Hb As shown in Fig. 4, the FTIR absorption spectrum of bovine Hb is similar to a great extent to that of human one (Liu et al., 2003). It contains two major bands: the first exists at 1655 cm1 arising from the amide CQO stretching (the amide I band), and another band lies approximately at 1540 cm1 originating from the N–H bending (the amide II band) vibrations in peptide groups in proteins. The bands at 1454 and 1388 cm1 originate from the bending vibrations of –CH2 and –CH3 groups of amino acids in the protein side chains. The –SH group vibrations
The EPR spectrum of 743 Gy gamma irradiated sample is shown in Fig. 2; from the figure it is clear that no new radicals have emerged, and no remarkable changes in the intensity of the first two signals were recorded. The unchanged intensity of the first signal (with all the irradiation doses) suggests no net change in MetHb, which may be explained as follows: The manipulated sample is in powder form, so any enzymatic reaction that can lead to change in MetHb content is excluded. The presence of oxygen is mandatory for MetHb production by other pathways such as the oxidation of heme iron by the electron transfer from Fe(II) to O2 creating Fe(III) and superoxide radicals (O 2 ) (Misra and Fridovich, 1972) During irradiation, wrapped samples were prevented from molecular oxygen in air. So, MetHb production through the second pathway is prevented; while the first pathway may be blocked by the removal of oxygen molecules from the bovine Hb sample during preparation in its powder form. The non-significant change in intensity of S2 by irradiation (Fig. 1), reflects the stability of non-heme iron content as radiation doses increase up to 743 Gy. It is clear from Fig. 1 that S3 suffered apparent significant decrease upon irradiation; which ensures the decrease of the net amount of hemichromes and reflects the crosslinking processes following Hb irradiation at high doses. Results showed that the most obvious radiation-induced change in bovine Hb EPR spectrum is the significant increase in S4 even for very low dose (4.95 Gy). This may be due to the increase in the production of free radicals in Hb protein (peroxyl and tyrosyl radicals) and reflects the high sensitivity of Hb protein to irradiation.
ARTICLE IN PRESS A.M. Maghraby, M.A. Ali / Radiation Physics and Chemistry 76 (2007) 1600–1605
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3.4. Radiation-induced changes in the Bovine Hb FTIR spectrum
3
3.5. Response to radiation Fig. 5 represents the response of the most distinctive two signals (S2 and S4) in EPR spectrum of bovine
Signal Intensity (arbitrary unit)
8000
6000
4000
Signal Intensity (arbitrary unit)
2
Fig. 4 shows the FTIR absorption spectrum of control and irradiated bovine Hb sample (743 Gy). Major changes induced by radiation involved marked decrease in intensity of several peaks as follows: The Tyrosine (Tyr) related band, found around 1517 cm1, is decreased in intensity (about 4.9%) after irradiation to about 743 Gy of Cs-137 gamma radiation. This means the deterioration of tyrosine molecular consistence, which confirms EPR spectroscopic analysis. Moreover, most of other bands showed decrease in intensity: marked decrease in the amide I band (about 5.78%) upon irradiation (743 Gy of Cs-137 gamma radiation) may be attributed to a change in a-helix or in the parallel or the anti-parallel b-sheets (Marta et al., 1996). The –SH stretching vibration around 2550 cm1, originates from the thiols of cysteine residues. The intensity of this band has decreased after irradiation to about 743 Gy, which reflects the change in a-104 cystein and b-93 cystein (Liu et al., 2003). In general, major changes in IR spectra upon irradiation reflect the reduction in a-helices contents, also the decrease in consistent tyrosine content. These changes are slightly different from the changes resulting in some Hb diseases, hence may reflect the impaired function of Hb molecules and hence its denaturation (Liu et al., 2003).
100000 9 8 7 6 5 4 3
2 10
100
1000
10000
100000
1000000
Dose (Gy)
Fig. 6. The response of S4 to ionizing gamma radiation (Co-60).
Hb to ionizing gamma radiation (Cs-137) from 4.95 to 743.14 Gy of Cs-137. From the figure it is clear that S2 has no response with radiation in this range and this agrees well with other published data (Zijlstra and Buursma, 1997). This radioresistance could be explained by an intrinsic crystalline property of porphyric ring, probably related to its structural rigidity (Zijlstra and Buursma, 1997). On the other side, S4 responses linearly to gamma radiation doses, its regression follows the linear relationship: Y ¼ 5.3 X+3531.4, with coefficient of determination (R2) ¼ 0.996. No new radicals have emerged when bovine Hb samples were irradiated to doses up to 300 kGy using Co-60 gamma radiation. The response of S4 to ionizing gamma radiation (Co-60) doses from 40 Gy to 300 kGy is plotted in Fig. 6. The fitting line power equation is: log(Y) ¼ B log(X)+A, where A ¼ 9.95, and B ¼ 0.187, with a coefficient of determination (R2) ¼ 0.993. The linear response of S4 reflects its non-saturating behavior at least up to 300 kGy which is a high enough limit for several applications. 4. Conclusion
2000
0 0
200
400
600
800
Dose (Gy) Fig. 5. Response of the most distinctive two signals (S2 and S4) in EPR spectrum of bovine Hb to ionizing gamma radiation (Cs-137).
Gamma irradiation of bovine Hb samples causes conformational changes in Hb molecular structure due to the non-saturating increase in tyrosine radicals production and the change in a-helices contents, which may cause the change in Hb functions performance. Results showed that the combined application of EPR and FTIR spectroscopy is a powerful tool for determining structural modification of Hb samples exposed to gamma irradiation. Further, detailed studies may be required for investigating spectroscopically the effect of ionizing radiation on patients receiving radiation during therapy sessions.
ARTICLE IN PRESS A.M. Maghraby, M.A. Ali / Radiation Physics and Chemistry 76 (2007) 1600–1605
References Comporti, M., Signorini, C., Buonocore, G., Ciccoli, L., 2002. Iron release, oxidative stress and erythrocyte aging. Free Radical Biol. Med. 32, 568–576. Dreval’, V.I., Sichevskaia, L.V., 2000. Exposure to ionizing radiation decreases hemoglobin binding to erythrocyte membrane. Biofizika 45 (6), 1086–1088. Farley, E., Florin, V., Lani, I., Houghton, A., Jaishanker, N., Halpern, H., Sutton, H., Srinivasan, V., 1995. Analysis of weekly complete blood counts in patients receiving standard fractionated partial body radiation therapy. Int. J. Radiat. Oncol. Biol. Phys. 33 (3), 607–617. Geoffrey, P., Jacobs, 1998. A review on the effects of ionizing radiation on blood and blood components. Radiat. Phys. Chem. 53 (5), 511–523. Gunther, M.R., Kelman, D.J., Corbett, J.T., Mason, R.P., 1995. Selfperoxidation of methmyoglobin results in formation of an oxygenreactive tryptophan-centered radical. J. Biol. Chem. 270, 16075–16081. http://www.sigmaaldrich.com/catalog/search/ProductDetail/FLUKA/ 51292. http://www.bmb.uga.edu/wampler/tutorial/prot4.html. Ikeya, M., 1993. New Applications of Electron Spin Resonance-Dating, Dosimetry and Microscopy. World Scientific, Singapore, pp. 383–384. Kelman, D.J., DeGraz, J.A., Mason, R.P., 1994. Reaction of myoglobin with hydrogen peroxide forms a peroxyl radical which oxidizes substrates. J. Biol. Chem. 269, 7458–7463. Kneipp, J., Balakrishnan, G., Chen, R., 2006. Dynamics of allostery in hemoglobin: roles of the penultimate tyrosine H bonds. J. Mol. Biol. 356, 335–353. Liu, K., Tsang, K., Li, C., Shaw, A., Mantsch, H., 2003. Infrared spectroscopic identification of b-thalassemia. Clin. Chem. 49 (7), 1125–1132. Marta, M., Patamia, M., Lupi, A., Antenucci, M., 1996. Bovine hemoglobin cross-linked through the b-chains, functional and structural aspects. J. Biol. Chem. 271 (13), 7473–7478. Miki, T., Kai, A., Ikeya, M., 1987. Electron spin resonance of blood stains and its application to the estimation of time after bleeding. Forensic Sci. Int. 35, 149–158. Misra, H., Fridovich, I., 1972. The generation of peroxide radical during the autoxidation of hemoglobin. J. Biol. Chem. 247, 6960–6962. Montebello, J., Wang, J.Z., Mayr, N.A., Yuh, W.T., Grecula, J.C., Nag, S., 2005. Longitudinal assessment of hemoglobin during chemo/ radiation therapy for cervical cancer. Int. J. Radiat. Oncol. Biol. Phys. 63 (1), S350. Mullon, J., Giacoppe, G., Clagett, C., 2000. Transfusions of polymerized bovine hemoglobin in a patient with sever hemolytic anemia. New Engl. J. Med. 342 (22), 1638–1643.
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Pelletier, J., Faivere, B., Touzery, C., Milan, C., Gilliard, P., 1978. Electron paramagnetic resonance studies of ferric hemoglobin before and after the action of ionizing radiations. Appl. Radiat. Isot. 29, 401–403. Puchala, M., Szweda-Lewandowska, Z., Kiefer, J., 2004. The influence of radiation quality on radiation-induced hemolysis and hemoglobin oxidation of human erythrocytes. J. Radiat. Res. (Tokyo) 45 (2), 275–279. Rachmilewitz, E., Peisach, J., Blumberg, W., 1971. Studies on the stability of oxyhemoglobin A and its constituent chains and their derivatives. J. Biol. Chem. 246, 3356–3366. S´lawska-Waniewska, A., Msiniewicz-Szablewska, E., Nedelko, N., Galazka-Friedman, J., Friedman, A., 2004. Magnetic studies of iron-entities in human tissues. J. Magn. Magn. Mater. 272–276, 2417–2419. Svistunenko, D.A., 2005. Reaction of haem containing proteins and enzymes with hydroperoxides: the radical view. Biochem. Biophys. Acta (Bioenerg.). 1707, 127–155. Svistunenko, D.A., Nathan, A.D., Wilson, M.T., Stidwill, R.P., Singer, M., Cooper, C.E., 1997a. Free radical in blood: a measure of hemoglobin autoxidation in vivo. J. Chem. Soc. Perkin Trans. 2, 2539–2543. Svistunenko, D.A., Rakesh, P., Voloshchenko, S.V., Wilson, M.T., 1997b. The globin-based free radical of ferryl hemoglobin is detected in normal human blood. J. Biol. Chem. 272, 7114–7121. Svistunenko, D.A., Dunne, J., Fryer, M., Nicholls, P., Reeder, B., Wilson, M., Bigott, M., Cutruzzola`, F., Cooper, C., 2002. Comparative study of tyrosine radicals in hemoglobin and myoglobins treated with hydrogen peroxide. Biophys. J. 49, 2845–2855. Svistunenko, D.A., Chris, E., Cooper, P., 2004. A new method of identifying the site of tyrosyl radicals in proteins. Biophys. J. 87, 582–595. Teicher, B.A., Wong, J.S., Takeuchi, H., Gravelin, L.M., Ara, G., Buxton, D., 1998. Allosteric effectors of hemoglobin as modulators of chemotherapy and radiation therapy in vitro and in vivo. Cancer Chemother. Pharmacol. 42 (1), 24–30. Venkatesh, B., Ramasamy, S., Asokan, R., Manoharan, P., 1997. Hemichromes in hemoglobin—an EPR study. J. Inorg. Biochem. 67, 121. Wajnberg, E., Bemski, G., 1993. Electron spin resonance measurements of erythrocytes and hemoglobin stored at 77 K. Naturwissenschaften 80, 472–473. Zijlstra, W.G., Buursma, A., 1997. Spectrophotometry of hemoglobin: absorption spectra of bovine oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, and methemoglobin. Comp. Biochem. Physiol. 118B, 743–749.
Radiation Physics and Chemistry 76 (2007) 1600–1605 www.elsevier.com/locate/radphyschem
Spectroscopic study of gamma irradiated bovine hemoglobin Ahmed Mohamed Maghrabya,, Maha Anwar Alib a
Radiation Dosimetry Department, National Institute of Standards (NIS), NIS-Tersa Street 12211, Haram-Giza, P.O. Box 136, Egypt b Biophysics Department, Faculty of Science, Cairo University, Giza, P.O. Box 12613, Egypt Received 14 November 2006; accepted 21 January 2007
Abstract In the present study, the effects of ionizing radiation of Cs-137 and Co-60 from 4.95 to 743.14 Gy and from 40 Gy to 300 kGy, respectively, on some bovine hemoglobin characteristics were studied. Such an effect was evaluated using electron paramagnetic resonance (EPR) spectroscopy, and infra-red (IR) spectroscopy. Bovine hemoglobin EPR spectra were recorded and analyzed before and after irradiation and changes were explained in detail. IR spectra of unirradiated and irradiated Bovine hemoglobin were recorded and analyzed also. It was found that ionizing radiation may lead to the increase of free radicals production, the decrease in a-helices contents, which reflects the degradation of hemoglobin molecular structure, or at least its incomplete performance. Results also show that the combined application of EPR and FTIR spectroscopy is a powerful tool for determining structural modification of bovine hemoglobin samples exposed to gamma irradiation. r 2007 Elsevier Ltd. All rights reserved. Keywords: EPR; ESR; Radiation; Bovine; Hemoglobin
1. Introduction The biological effects of radiation could be analyzed in part by the study of the primary processes of radiationinduced damage of vitally important proteins. Hemoglobin (Hb) is the important iron-containing oxygen transport protein in the body; therefore, radiation-induced damage of Hb is a subject of various studies (Teicher et al., 1998; Geoffrey and Jacobs, 1998; Dreval’ and Sichevskaia, 2000; Puchala et al., 2004; Montebello et al., 2005). The study of the effect of ionizing radiation on the molecular structure of Hb is quite important for several reasons; one of them is to investigate the effect of probable changes in Hb molecules after radiation therapy sessions or even in cases of diagnostic applications. It is important also for occupational radiation workers and in emergency or accident cases, where the use of complete blood count (CBC) does not indicate the functionality of Hb content while it may possess normal count (Farley et al., 1995). Also, the study of some properties of heme may be an Corresponding author. Tel.: +2 0124509723; fax: +2 023867451.
E-mail address: [email protected] (A.M. Maghraby). 0969-806X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2007.01.008
useful indicator for its denaturation (Wajnberg and Bemski, 1993). For erythrocyte proteins, Hb normally account for about 90% and spectrin for about 10%. Comporti et al. (2002) showed that Hb denaturation occurred after irradiation and this protein is the most susceptible to reactive oxygen species attacks. Although it was studied before (Pelletier et al., 1978), the action of ionizing radiation on Hb was not reported quantitatively (at the knowledge of authors), and the limit and the kind of the probable effect(s) were not provided accurately, hence there is a need to determine such effect(s) quantitatively and accurately. Therefore, the aim of the present in vitro study was to study the effect of ionizing radiation on Hb molecule itself and eliminating other contributing factors that may lead to the enhancement or suppression of this effect. Spectrophotometric properties of bovine Hb are only slightly different from those of human blood (Zijlstra and Buursma, 1997). Its importance may be reflected by the interest in the development of safe, and effective substitute for human erythrocytes as a transfusible medium for oxygen transport. Polymerized forms of bovine Hb, such as
ARTICLE IN PRESS A.M. Maghraby, M.A. Ali / Radiation Physics and Chemistry 76 (2007) 1600–1605
1601
HBOC-201, show particular promise. They have a molecular structure similar to that of human Hb but have lower concentrations of organic phosphates, resulting in more pronounced oxygen unloading in ischemic tissue and increased Hb binding of carbon dioxide in the deoxygenated state (Mullon et al., 2000). Moreover, some foods (especially children foods) may contain bovine Hb concentrate, designed to provide certain quantity of bio-available iron per day (Zijlstra and Buursma, 1997), hence this food (or its components) may require doses of about 30–50 kGy for industrial sterilization. Therefore, in the present study the effect of ionizing radiation of Cs-137 and Co-60 from 4.95 to 743.14 Gy (clinical usages) and from 40 Gy to 300 kGy (industrial use), respectively, on some bovine Hb characteristics was studied. 2. Materials and methods 2.1. Irradiation facilities Cesium-137 gamma source (model: GB-150-Canada), was used for samples irradiation. It was manufactured by Atomic Energy of Canada Limited on April 1970 with original activity of 1000 Ci (3.7 1010 kBq), dose rate were about 1.2 Gy/h at 1 m. Although the half-life time of the source is not short (30.17 a), doses were corrected for source decay each irradiation turn. Irradiation was also performed using a Co-60 g-source (irradiation cell) installed at the National Center for Radiation Research and Technology (NCRRT) at the Egyptian Atomic Energy Authority. It possesses activity of 50,000 Ci, with dose rate of about 4.0 kGy/h. Samples were irradiated at room temperature in a Perspex phantom irradiation capsules.
Fig. 1. Bovine Hb is composed of two pairs of non-identical subunits, alpha and beta. Each alpha–beta pair is more closely associated than they are with each other, but the overall arrangement is roughly tetrahedral.
methemoglobin (http://www.sigmaaldrich.com/catalog/search/ ProductDetail/FLUKA/51292). Structure of bovine Hb is shown in Fig. 1. It is composed of two pairs of non-identical subunits, alpha and beta. Each alpha–beta pair is more closely associated than they are with each other, but the overall arrangement is roughly tetrahedral (http://www.bmb.uga.edu/ wampler/tutorial/prot4.html) (Marta et al., 1996). Powder Hb samples were pressed into disks and then were wrapped in thin-sealed plastic bags prior to irradiation in order to minimize the possibility of interaction between samples and molecular oxygen. 2.4. Evaluation method of EPR spectra
2.2. Radiation dose determination Air kerma (Kair) was determined according to the International Atomic Energy Agency (IAEA) code of practice TRS-(381) (IAEA, 1997). Kair determination was performed using the secondary standard dosimetry system of the National Institute of Standards (NIS), Egypt, which is composed of a NPL-NE2560 electrometer (UK) and a NE2561 ionization chamber (UK). The secondary standard calibration system was calibrated at the Bureau International des Poids et Mesure (BIPM). The expanded uncertainty associated to Kair determination was about 0.9% at 95% level of confidence (coverage factor ¼ 2). 2.3. Bovine Hb samples Powder bovine Hb of average molecular weight about 64,500 was obtained from Sigma (St. Louis, MO, USA) and used without further purification. Since native Hb is readily oxidized in air, this preparation may be predominantly
The EPR system used in this study is an EMXBRUKER system, Germany, supplied by a 9.5 GHz microwave (X-band) Gunn-Oscillator Bridge with automatic tuning capability and a rectangular 4102 ST cavity operating in the TE102 mode. The standard EMX Signal Channel can be operated at any modulation frequency between 6 and 100 kHz, and has unsurpassed phase resolution and stability. EPR spectra of the empty tubes were recorded before recording the samples spectra in order to ensure the purity of the obtained signals. Readings were corrected to the peak-to-peak height of the reference standard material (Pyrolyzed sucrose) in order to correct for the change in the spectrometer sensitivity. Sample weight was about 72.00712 mg and sample mass normalization was performed for each acquired signal intensity. The spectrum of each single sample was recorded three or more successive times. All measurements were performed at room temperature.
ARTICLE IN PRESS A.M. Maghraby, M.A. Ali / Radiation Physics and Chemistry 76 (2007) 1600–1605
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Parameters used for spectra acquisition are as follows: modulation amplitude is 2 mT, modulation frequency is 100 kHz, microwave power is 1.586 mW, center field is 348 mT, sweep width is 20 mT, time constant is 81.92, and conversion time is 40.96 ms for 1024 data points resulting in sweep time about 42 s. 2.5. FTIR spectra acquisition FTIR spectra were recorded using a double-beam IR spectrometer of type Jasco-460 FTIR plus, Japan. Each spectrum is recorded over wavenumber range 4000– 400 cm1. Hb sample is mixed intimately with finely powdered KBr and the mixture is squeezed in a press to about 1000 atm. Under these conditions, the KBr becomes glassy and forms a thin translucent disk in which the finely ground sample is suspended. 3. Results and discussion 3.1. Bovine Hb EPR spectral features Fig. 2 represents the EPR spectra of unirradiated (solid line) and 743 Gy gamma irradiated sample (dotted line). Major features of Bovine Hb EPR spectrum are comparable to the human one (Ikeya, 1993). About four features comprise the spectrum of g-factor equal to 5.910170.0860, 4.275070.0031, 2.147370.0227 and 2.005570.0010, respectively. The first two signals are due to Fe (III) of high spin form (S ¼ 5/2). The first signal (S1) is associated with oxidized heme iron, which clarifies its indication to methemoglobin (MetHb), in which a water molecule replaces O2 ion as a ligand of iron (Wajnberg and Bemski 1993). The second signal (S2) is corresponding to non-heme Fe(III) ions at sites endowed with rhombic symmetry, which is not associated with species involved in blood, such as transferrin which causes an EPR signal near g ¼ 4.3
(S´lawska-Waniewska et al., 2004). From Fig. 2, it is clear that S2 is greater than S1, while this is not true in case of human Hb spectrum (Ikeya, 1993). This means higher nonheme iron content in bovine Hb than that of human. The nature of bovine Hb itself or the deterioration of its molecular structure and hence the decomposition of heme during sample preparation may give reason for the increase of non-heme iron content. Third signal group (S3) is associated with low spin derivatives of ferrihemoglobin called ‘‘hemichrome’’, copper proteins and some transition-metal complexes (Rachmilewitz et al., 1971). Hemichromes are low spin derivatives of ferric Hb brought about through discrete reversible or irreversible changes of protein conformation (Venkatesh et al., 1997). The purity of our sample is about 95%, hemichromes are not easily to be separated from Hb molecules but the rest which is about 5% may contain some of these hemichromes that produce S3. Changes in normal Hb under the effect of time, pH and protein denaturants such as urea or salicylate can form different kinds of hemichromes with different endogenous ligands; hence hemichromes form the primary step to the destructive pathway for denaturation (Venkatesh et al., 1997). With regard to the fourth signal (S4), it appears as a singlet with no hyperfine structures as shown in Fig. 2. This signal is attributed to free radicals in hemoglobin formed by the degradation of blood constituents (Miki et al., 1987). Several investigators agree that at least two different kinds of radicals are formed on the protein (Kelman et al., 1994; Gunther et al., 1995). Although the formation of peroxyl radicals is well proven (Ikeya, 1993; Miki et al., 1987), this kind of radical constitutes only a fraction of the total concentration of radicals (Svistunenko et al., 1997a, b). The globin-based free radical (HB(Fe(IV)QO)) was suggested to be major contributor for S4 (Svistunenko et al., 1997a,b). Many investigations revealed that it is the
Fig. 2. EPR spectra of unirradiated and 743 Gy gamma irradiated bovine Hb samples recorded at room temperature.
ARTICLE IN PRESS A.M. Maghraby, M.A. Ali / Radiation Physics and Chemistry 76 (2007) 1600–1605
Fig. 3. Radiation-induced Tyrosine (Tyr) radical in the bovine Hb (from Svistunenko et al., 2002).
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from the cystein residues appear around 2550 cm1. The bands between 2872 and 3000 cm1 are the symmetric and asymmetric –CH2 and –CH3 stretching vibrations from protein side chains. Other protein bands are the amide B band at about 3050 cm1 and amide A at about 3309 cm1. The amide band most widely used in studies of protein secondary structure is the amide I mode, which has been used extensively to quantify a-helices, b-sheets, turns, and non-ordered structures in proteins. Overlapping components in the amide I band includes ahelix (1657 cm1) or parallel and antiparallel b-sheets (1640 and 1680 cm1, respectively). The vibration of the tyrosine ring (about 1517 cm1) originates from the side chains of the protein overlaps in the amide II band (Gunther et al., 1995). The tyrosine residues adjacent to the C termini of Hb subunits are expected to play important structural roles, because the C termini are the loci of T-state quaternary salt-bridges, and because the tyrosine side chains bridge the H and F helices via H bonds to the carbonyl groups (Kneipp et al., 2006). 3.3. Radiation-induced changes in the bovine Hb EPR spectra
Fig. 4. IR spectra of unirradiated and 743 Gy gamma irradiated bovine Hb samples, recorded at room temperature.
tyrosine (Tyr) radical (shown in Fig. 3) (Svistunenko et al., 1997a,b, 2002, 2004; Svistunenko, 2005) and showed the resemblance between this signal and that of the formed radical after addition of hydrogen peroxide to the met form of Hb: FeIII
R + H2O2
FeIV = OR+ + H2O
(1)
3.2. FTIR spectral features of bovine Hb As shown in Fig. 4, the FTIR absorption spectrum of bovine Hb is similar to a great extent to that of human one (Liu et al., 2003). It contains two major bands: the first exists at 1655 cm1 arising from the amide CQO stretching (the amide I band), and another band lies approximately at 1540 cm1 originating from the N–H bending (the amide II band) vibrations in peptide groups in proteins. The bands at 1454 and 1388 cm1 originate from the bending vibrations of –CH2 and –CH3 groups of amino acids in the protein side chains. The –SH group vibrations
The EPR spectrum of 743 Gy gamma irradiated sample is shown in Fig. 2; from the figure it is clear that no new radicals have emerged, and no remarkable changes in the intensity of the first two signals were recorded. The unchanged intensity of the first signal (with all the irradiation doses) suggests no net change in MetHb, which may be explained as follows: The manipulated sample is in powder form, so any enzymatic reaction that can lead to change in MetHb content is excluded. The presence of oxygen is mandatory for MetHb production by other pathways such as the oxidation of heme iron by the electron transfer from Fe(II) to O2 creating Fe(III) and superoxide radicals (O 2 ) (Misra and Fridovich, 1972) During irradiation, wrapped samples were prevented from molecular oxygen in air. So, MetHb production through the second pathway is prevented; while the first pathway may be blocked by the removal of oxygen molecules from the bovine Hb sample during preparation in its powder form. The non-significant change in intensity of S2 by irradiation (Fig. 1), reflects the stability of non-heme iron content as radiation doses increase up to 743 Gy. It is clear from Fig. 1 that S3 suffered apparent significant decrease upon irradiation; which ensures the decrease of the net amount of hemichromes and reflects the crosslinking processes following Hb irradiation at high doses. Results showed that the most obvious radiation-induced change in bovine Hb EPR spectrum is the significant increase in S4 even for very low dose (4.95 Gy). This may be due to the increase in the production of free radicals in Hb protein (peroxyl and tyrosyl radicals) and reflects the high sensitivity of Hb protein to irradiation.
ARTICLE IN PRESS A.M. Maghraby, M.A. Ali / Radiation Physics and Chemistry 76 (2007) 1600–1605
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3.4. Radiation-induced changes in the Bovine Hb FTIR spectrum
3
3.5. Response to radiation Fig. 5 represents the response of the most distinctive two signals (S2 and S4) in EPR spectrum of bovine
Signal Intensity (arbitrary unit)
8000
6000
4000
Signal Intensity (arbitrary unit)
2
Fig. 4 shows the FTIR absorption spectrum of control and irradiated bovine Hb sample (743 Gy). Major changes induced by radiation involved marked decrease in intensity of several peaks as follows: The Tyrosine (Tyr) related band, found around 1517 cm1, is decreased in intensity (about 4.9%) after irradiation to about 743 Gy of Cs-137 gamma radiation. This means the deterioration of tyrosine molecular consistence, which confirms EPR spectroscopic analysis. Moreover, most of other bands showed decrease in intensity: marked decrease in the amide I band (about 5.78%) upon irradiation (743 Gy of Cs-137 gamma radiation) may be attributed to a change in a-helix or in the parallel or the anti-parallel b-sheets (Marta et al., 1996). The –SH stretching vibration around 2550 cm1, originates from the thiols of cysteine residues. The intensity of this band has decreased after irradiation to about 743 Gy, which reflects the change in a-104 cystein and b-93 cystein (Liu et al., 2003). In general, major changes in IR spectra upon irradiation reflect the reduction in a-helices contents, also the decrease in consistent tyrosine content. These changes are slightly different from the changes resulting in some Hb diseases, hence may reflect the impaired function of Hb molecules and hence its denaturation (Liu et al., 2003).
100000 9 8 7 6 5 4 3
2 10
100
1000
10000
100000
1000000
Dose (Gy)
Fig. 6. The response of S4 to ionizing gamma radiation (Co-60).
Hb to ionizing gamma radiation (Cs-137) from 4.95 to 743.14 Gy of Cs-137. From the figure it is clear that S2 has no response with radiation in this range and this agrees well with other published data (Zijlstra and Buursma, 1997). This radioresistance could be explained by an intrinsic crystalline property of porphyric ring, probably related to its structural rigidity (Zijlstra and Buursma, 1997). On the other side, S4 responses linearly to gamma radiation doses, its regression follows the linear relationship: Y ¼ 5.3 X+3531.4, with coefficient of determination (R2) ¼ 0.996. No new radicals have emerged when bovine Hb samples were irradiated to doses up to 300 kGy using Co-60 gamma radiation. The response of S4 to ionizing gamma radiation (Co-60) doses from 40 Gy to 300 kGy is plotted in Fig. 6. The fitting line power equation is: log(Y) ¼ B log(X)+A, where A ¼ 9.95, and B ¼ 0.187, with a coefficient of determination (R2) ¼ 0.993. The linear response of S4 reflects its non-saturating behavior at least up to 300 kGy which is a high enough limit for several applications. 4. Conclusion
2000
0 0
200
400
600
800
Dose (Gy) Fig. 5. Response of the most distinctive two signals (S2 and S4) in EPR spectrum of bovine Hb to ionizing gamma radiation (Cs-137).
Gamma irradiation of bovine Hb samples causes conformational changes in Hb molecular structure due to the non-saturating increase in tyrosine radicals production and the change in a-helices contents, which may cause the change in Hb functions performance. Results showed that the combined application of EPR and FTIR spectroscopy is a powerful tool for determining structural modification of Hb samples exposed to gamma irradiation. Further, detailed studies may be required for investigating spectroscopically the effect of ionizing radiation on patients receiving radiation during therapy sessions.
ARTICLE IN PRESS A.M. Maghraby, M.A. Ali / Radiation Physics and Chemistry 76 (2007) 1600–1605
References Comporti, M., Signorini, C., Buonocore, G., Ciccoli, L., 2002. Iron release, oxidative stress and erythrocyte aging. Free Radical Biol. Med. 32, 568–576. Dreval’, V.I., Sichevskaia, L.V., 2000. Exposure to ionizing radiation decreases hemoglobin binding to erythrocyte membrane. Biofizika 45 (6), 1086–1088. Farley, E., Florin, V., Lani, I., Houghton, A., Jaishanker, N., Halpern, H., Sutton, H., Srinivasan, V., 1995. Analysis of weekly complete blood counts in patients receiving standard fractionated partial body radiation therapy. Int. J. Radiat. Oncol. Biol. Phys. 33 (3), 607–617. Geoffrey, P., Jacobs, 1998. A review on the effects of ionizing radiation on blood and blood components. Radiat. Phys. Chem. 53 (5), 511–523. Gunther, M.R., Kelman, D.J., Corbett, J.T., Mason, R.P., 1995. Selfperoxidation of methmyoglobin results in formation of an oxygenreactive tryptophan-centered radical. J. Biol. Chem. 270, 16075–16081. http://www.sigmaaldrich.com/catalog/search/ProductDetail/FLUKA/ 51292. http://www.bmb.uga.edu/wampler/tutorial/prot4.html. Ikeya, M., 1993. New Applications of Electron Spin Resonance-Dating, Dosimetry and Microscopy. World Scientific, Singapore, pp. 383–384. Kelman, D.J., DeGraz, J.A., Mason, R.P., 1994. Reaction of myoglobin with hydrogen peroxide forms a peroxyl radical which oxidizes substrates. J. Biol. Chem. 269, 7458–7463. Kneipp, J., Balakrishnan, G., Chen, R., 2006. Dynamics of allostery in hemoglobin: roles of the penultimate tyrosine H bonds. J. Mol. Biol. 356, 335–353. Liu, K., Tsang, K., Li, C., Shaw, A., Mantsch, H., 2003. Infrared spectroscopic identification of b-thalassemia. Clin. Chem. 49 (7), 1125–1132. Marta, M., Patamia, M., Lupi, A., Antenucci, M., 1996. Bovine hemoglobin cross-linked through the b-chains, functional and structural aspects. J. Biol. Chem. 271 (13), 7473–7478. Miki, T., Kai, A., Ikeya, M., 1987. Electron spin resonance of blood stains and its application to the estimation of time after bleeding. Forensic Sci. Int. 35, 149–158. Misra, H., Fridovich, I., 1972. The generation of peroxide radical during the autoxidation of hemoglobin. J. Biol. Chem. 247, 6960–6962. Montebello, J., Wang, J.Z., Mayr, N.A., Yuh, W.T., Grecula, J.C., Nag, S., 2005. Longitudinal assessment of hemoglobin during chemo/ radiation therapy for cervical cancer. Int. J. Radiat. Oncol. Biol. Phys. 63 (1), S350. Mullon, J., Giacoppe, G., Clagett, C., 2000. Transfusions of polymerized bovine hemoglobin in a patient with sever hemolytic anemia. New Engl. J. Med. 342 (22), 1638–1643.
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Pelletier, J., Faivere, B., Touzery, C., Milan, C., Gilliard, P., 1978. Electron paramagnetic resonance studies of ferric hemoglobin before and after the action of ionizing radiations. Appl. Radiat. Isot. 29, 401–403. Puchala, M., Szweda-Lewandowska, Z., Kiefer, J., 2004. The influence of radiation quality on radiation-induced hemolysis and hemoglobin oxidation of human erythrocytes. J. Radiat. Res. (Tokyo) 45 (2), 275–279. Rachmilewitz, E., Peisach, J., Blumberg, W., 1971. Studies on the stability of oxyhemoglobin A and its constituent chains and their derivatives. J. Biol. Chem. 246, 3356–3366. S´lawska-Waniewska, A., Msiniewicz-Szablewska, E., Nedelko, N., Galazka-Friedman, J., Friedman, A., 2004. Magnetic studies of iron-entities in human tissues. J. Magn. Magn. Mater. 272–276, 2417–2419. Svistunenko, D.A., 2005. Reaction of haem containing proteins and enzymes with hydroperoxides: the radical view. Biochem. Biophys. Acta (Bioenerg.). 1707, 127–155. Svistunenko, D.A., Nathan, A.D., Wilson, M.T., Stidwill, R.P., Singer, M., Cooper, C.E., 1997a. Free radical in blood: a measure of hemoglobin autoxidation in vivo. J. Chem. Soc. Perkin Trans. 2, 2539–2543. Svistunenko, D.A., Rakesh, P., Voloshchenko, S.V., Wilson, M.T., 1997b. The globin-based free radical of ferryl hemoglobin is detected in normal human blood. J. Biol. Chem. 272, 7114–7121. Svistunenko, D.A., Dunne, J., Fryer, M., Nicholls, P., Reeder, B., Wilson, M., Bigott, M., Cutruzzola`, F., Cooper, C., 2002. Comparative study of tyrosine radicals in hemoglobin and myoglobins treated with hydrogen peroxide. Biophys. J. 49, 2845–2855. Svistunenko, D.A., Chris, E., Cooper, P., 2004. A new method of identifying the site of tyrosyl radicals in proteins. Biophys. J. 87, 582–595. Teicher, B.A., Wong, J.S., Takeuchi, H., Gravelin, L.M., Ara, G., Buxton, D., 1998. Allosteric effectors of hemoglobin as modulators of chemotherapy and radiation therapy in vitro and in vivo. Cancer Chemother. Pharmacol. 42 (1), 24–30. Venkatesh, B., Ramasamy, S., Asokan, R., Manoharan, P., 1997. Hemichromes in hemoglobin—an EPR study. J. Inorg. Biochem. 67, 121. Wajnberg, E., Bemski, G., 1993. Electron spin resonance measurements of erythrocytes and hemoglobin stored at 77 K. Naturwissenschaften 80, 472–473. Zijlstra, W.G., Buursma, A., 1997. Spectrophotometry of hemoglobin: absorption spectra of bovine oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, and methemoglobin. Comp. Biochem. Physiol. 118B, 743–749.