Spectrophotometry of Hemoglobin: Absorption Spectra of Bovine Oxyhemoglobin, Deoxyhemoglobin, Carboxyhemoglobin, and Methemoglobin

Zijlstra, W. G.; Buursma, Anneke

doi:10.1016/s0305-0491(97)00230-7

Cited by 214 publications

(193 citation statements)

References 12 publications

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“…Where needed the Hb was reduced by adding a slight molar excess of sodium dithionite and dialysing against N 2 -equilibrated 0.01moll -1 buffer. Absorption spectra of fully reduced, oxygenated Hbs showed / peak (A576.0nm/A539.7nm) absorption ratios of 1.06 as characterizes pure human oxyHb (Zijlstra and Buursma, 1997). O 2 equilibria were determined in 0.1moll -1 Hepes buffer using a modified diffusion chamber and Wösthoff gas mixing pumps as previously described (Weber, 1981;Weber et al, 2000).…”

Section: Methodsmentioning

confidence: 99%

ATP-induced temperature independence of hemoglobin–O2 affinity in heterothermic billfish

Weber

Campbell

Fago

et al. 2010

Journal of Experimental Biology

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SUMMARYThe inverse relationship between temperature and hemoglobin-O 2 affinity resulting from the exothermic nature of heme oxygenation favors O 2 unloading from blood to warm, metabolically active tissues. However, this temperature sensitivity is maladaptive, and commonly countered in regional heterotherms, where it may hamper unloading (e.g. in cold extremities of arctic mammals) or increase the diffusive arterio-venous short-circuiting of O 2 (e.g. in counter-current heat exchangers of warm swimming muscles of tuna). We hypothesized analogous blood specializations in heterothermic billfish, whose warm eyes and brains increase the temporal resolution of vision, and measured hemoglobin-O 2 binding properties in three species over a wide pH range, at two temperatures, and in the absence and presence of the major red cell effector, ATP, permitting detailed assessment of overall oxygenation enthalpies (⌬HЈ) and contributions from oxygenation-linked proton and ATP dissociation. . Thus in addition to allosterically modulating hemoglobin-O 2 affinity, ATP diminishes its temperature sensitivity, reducing deleterious arterio-venous short-circuiting of oxygen in the cranial billfish heat exchangers. The mechanism underlying this reduction in oxygenation enthalpy differs fundamentally from that in tuna, supporting independent evolution of this trait in these scombroid lineages.

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Section: Methodsmentioning

confidence: 99%

ATP-induced temperature independence of hemoglobin–O2 affinity in heterothermic billfish

Weber

Campbell

Fago

et al. 2010

Journal of Experimental Biology

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“…It is also shown that, in crystallo, a dominantly Fe visible data (Fig. 1A, Inset) (19). Typically, the rising-edge energy positions of transition metal K-edges have been used as signatures of the oxidation state of the absorbing metal center (20).…”

Section: Significancementioning

confidence: 99%

X-ray absorption spectroscopic investigation of the electronic structure differences in solution and crystalline oxyhemoglobin

Wilson

Green

Mathews

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Hemoglobin (Hb) is the heme-containing O 2 transport protein essential for life in all vertebrates. The resting high-spin (S = 2) ferrous form, deoxy-Hb, combines with triplet O 2 , forming diamagnetic (S = 0) oxy-Hb. Understanding this electronic structure is the key first step in understanding transition metal-O 2 interaction. However, despite intense spectroscopic and theoretical studies, the electronic structure description of oxy-Hb remains elusive, with at least three different descriptions proposed by Pauling, Weiss, and McClureGoddard, based on theory, spectroscopy, and crystallography. Here, a combination of X-ray absorption spectroscopy and extended X-ray absorption fine structure, supported by density functional theory calculations, help resolve this debate. X-ray absorption spectroscopy data on solution and crystalline oxy-Hb indicate both geometric and electronic structure differences suggesting that two of the previous descriptions are correct for the Fe-O 2 center in oxy-Hb. These results support the multiconfigurational nature of the ground state developed by theoretical results. Additionally, it is shown here that small differences in hydrogen bonding and solvation effects can tune the ground state, tipping it into one of the two probable configurations. These data underscore the importance of solution spectroscopy and show that the electronic structure in the crystalline form may not always reflect the true ground-state description in solution.H emoglobin (Hb) is the iron-containing heme protein essential for oxygen transport in all vertebrates. The protein is an assembly of four globular subunits, each containing a heme B (protoporphyrin IX group) (1). The first characterization of Hb was done in the mid-1800s (2, 3), with the magnetic properties first elucidated in early 1900s. Later, Pauling and Coryell (4) showed that whereas resting venous Hb is paramagnetic, the oxygenated arterial form (oxy-Hb) is diamagnetic in nature. The first structure determination (X-ray diffraction) was done by Perutz et al. (5) in 1960, who was awarded the Nobel prize for this work. In 1974, the geometry of O 2 binding was shown to be end-on η 1 -Fe-O 2 , based on X-ray diffraction data on a biomimetic model complex (6), which was later confirmed by diffraction data on oxyHb. However, the debate over the electronic structure of O 2 binding in Hb and the nature of the Fe-O 2 bond continues today.Although the diamagnetic nature of oxy-Hb has been described by several different models, three have persisted. Starting from deoxy-Hb (Fe 2+ , S = 5/2) and O 2 (neutral, S = 1) and using a valence bond theory, Pauling proposed that the oxy-Hb is a Fe 2+ , S = 0 system that accepts one lone pair from the sp 2 hybridized O 2 , forming a dative bond (Scheme 1) (7). The Weiss model involves one-electron reduction of O 2 by the ferrous heme in deoxy-Hb (8), which leads to antiferromagnetic coupling between the Fe 3+ (S = 1/2) and O In this study, quantitative Fe K-edge XAS and density functional theory (DFT) calculations have ...

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“…Cortical areas were substantially less pigmented for felids, canids and mice than for homologous sites in cetaceans, pinnipeds and sea otters. Spectrophotometric analyses of total pigmentation at the average absorbance peak wavelength of 558-568 nm for carboxy-Hb (Zijlstra & Buursma 1997) and deoxy-neuroglobin (Burmester et al 2000) revealed a 2.4-fold higher optical density for samples from marine species (0.599G0.078 OD, nZ10) compared with terrestrial species (0.245G0.022 OD, nZ5). Admittedly, several factors may contribute to visible staining, and therefore pigmentation, of the tissues including the postmortem interval, degree of erythrocyte disruption and blood vessel degradation.…”

Section: Resultsmentioning

confidence: 99%

“…Approximately 0.02 g of sodium sulphite was added to ensure complete reduction. The absorbance of each sample was read at room temperature on a desktop spectrophotometer (UV-visible Bio spec-1601, Shimadzu Corp., Kyoto, Japan) over the wavelength range 416-568 nm encompassing peak absorbencies for Hb (Zijlstra & Buursma 1997), cytoglobin (Fago et al 2004a) and neuroglobin (Burmester et al 2000;Dewilde et al 2001;Fago et al 2004a).…”

Section: Methodsmentioning

confidence: 99%

Running, swimming and diving modifies neuroprotecting globins in the mammalian brain

et al. 2007

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The vulnerability of the human brain to injury following just a few minutes of oxygen deprivation with submergence contrasts markedly with diving mammals, such as Weddell seals (Leptonychotes weddellii), which can remain underwater for more than 90 min while exhibiting no neurological or behavioural impairment. This response occurs despite exposure to blood oxygen levels concomitant with human unconsciousness. To determine whether such aquatic lifestyles result in unique adaptations for avoiding ischaemic-hypoxic neural damage, we measured the presence of circulating (haemoglobin) and resident (neuroglobin and cytoglobin) oxygen-carrying globins in the cerebral cortex of 16 mammalian species considered terrestrial, swimming or diving specialists. Here we report a striking difference in globin levels depending on activity lifestyle. A nearly 9.5-fold range in haemoglobin concentration (0.17-1.62 g Hb 100 g brain wet wt K1) occurred between terrestrial and deep-diving mammals; a threefold range in resident globins was evident between terrestrial and swimming specialists. Together, these two globin groups provide complementary mechanisms for facilitating oxygen transfer into neural tissues and the potential for protection against reactive oxygen and nitrogen groups. This enables marine mammals to maintain sensory and locomotor neural functions during prolonged submergence, and suggests new avenues for averting oxygen-mediated neural injury in the mammalian brain.

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Spectrophotometry of Hemoglobin: Absorption Spectra of Bovine Oxyhemoglobin, Deoxyhemoglobin, Carboxyhemoglobin, and Methemoglobin

Cited by 214 publications

References 12 publications

ATP-induced temperature independence of hemoglobin–O2 affinity in heterothermic billfish

ATP-induced temperature independence of hemoglobin–O2 affinity in heterothermic billfish

X-ray absorption spectroscopic investigation of the electronic structure differences in solution and crystalline oxyhemoglobin

Running, swimming and diving modifies neuroprotecting globins in the mammalian brain

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