Preparation and Properties of α- and β-Chains from Human Hemoglobin

Geraci, Giuseppe; Parkhurst, Lawrence J.; Gibson, Quentin H.

doi:10.1016/s0021-9258(18)93675-6

Cited by 360 publications

(57 citation statements)

References 15 publications

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“…Hb A hemolysate, prepared as described previously (Geraci et al 1969, was diluted to ϳ120 M (in heme) in buffer (0.1 M sodium phosphate, pH 7.3). The hemolysate contained less than 5% Hb A 2 impurity, as determined by ion exchange chromatography with a DE-52 matrix.…”

Section: Experimental Sample Preparation and Instrumentationmentioning

confidence: 99%

Multiple Geminate Ligand Recombinations in Human Hemoglobin

Esquerra

Goldbeck

Reaney

et al. 2000

Biophysical Journal

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The geminate ligand recombination reactions of photolyzed carbonmonoxyhemoglobin were studied in a nanosecond double-excitation-pulse time-resolved absorption experiment. The second laser pulse, delayed by intervals as long as 400 ns after the first, provided a measure of the geminate kinetics by rephotolyzing ligands that have recombined during the delay time. The peak-to-trough magnitude of the Soret band photolysis difference spectrum measured as a function of the delay between excitation pulses showed that the room temperature kinetics of geminate recombination in adult human hemoglobin are best described by two exponential processes, with lifetimes of 36 and 162 ns. The relative amounts of bimolecular recombination to T- and R-state hemoglobins and the temperature dependence of the submicrosecond kinetics between 283 and 323 K are also consistent with biexponential kinetics for geminate recombination. These results are discussed in terms of two models: geminate recombination kinetics modulated by concurrent protein relaxation and heterogeneous kinetics arising from alpha and beta chain differences.

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Section: Experimental Sample Preparation and Instrumentationmentioning

confidence: 99%

Multiple Geminate Ligand Recombinations in Human Hemoglobin

Esquerra

Goldbeck

Reaney

et al. 2000

Biophysical Journal

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“…Oxyhaemoglobin was converted to deoxyhaemoglobin either by exhaustively purging the solution with nitrogen and then adding a small amount of solid dithionite or by subjecting the solution to repeated freeze-pump-thaw cycles on a vacuum line. Carboxyhaemoglobin was prepared by bubbling carbon monoxide through oxyhaemoglobin solution for an extended period, a and |3 chains in the oxy form were separated as their mercury complexes by using the two column procedure of Geraci et al (Geraci, Parkhurst & Gibson 1969). The separated complexes were concentrated to above 5 x 10-4 m by vacuum dialysis to give samples suitable for subsequent e.s.r.…”

Section: E X P E R Im E N T a Lmentioning

confidence: 99%

Electron capture by oxyhaemoglobin: an e. s. r. study

Symons

Petersen

1978

Proc. R. Soc. Lond. B.

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Exposure of aqueous glasses containing oxyhaemoglobin to 60 Co γ rays at 77 K gave two similar paramagnetic centres whose electron spin resonance (e. s. r.) spectra resembled those of low-spin ferric derivatives. These were shown to be formed in the α and β chains by electron capture. The use of oxygen labelled with 17 O showed the presence of two inequivalent oxygen atoms and it is shown that the unpaired electron has considerable spin density on the dioxygen ligand as well as on iron. When warmed above 77 K two new paramagnetic centres were formed, possibly as a result of protonation, before the formation of normal high-spin methaemoglobin, presumably by loss of HO 2 ¯ . Oxymyoglobin gave comparable centres.

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“…The α and β globin subunits were isolated from each of the hemoglobin variants using previously published protocol 6,33,34 . Briefly, 100 mg PMB per 1 g of hemoglobin was dissolved in a minimum volume of 0.1 M KOH and 1 M acetic acid was added till very light precipitate persisted.…”

Section: Methodsmentioning

confidence: 99%

“…The α and β globin subunits were isolated from each of the hemoglobin variants using previously published protocol. 6,33,34 Briefly, 100 mg PMB per 1 g of hemoglobin was dissolved in a minimum volume of 0.1 M KOH and 1 M acetic acid was added till very light precipitate persisted. PMB solution was added to 50 mg/ml solution of hemoglobin in 10 mM Tris-HCl 200 mM NaCl, pH 8.0, and final pH was adjusted to 6.0 by addition of 1 M acetic acid and mixture was incubated at 4 C for 12 hr.…”

Section: Isolation and Purification Of α And β Globin Subunits From H...mentioning

confidence: 99%

Erythroid spectrin binding modulates peroxidase and catalase activity of heme proteins

et al. 2022

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Hemoglobin oxidation due to oxidative stress and disease conditions leads to the generation of ROS (reactive oxygen species) and membrane attachment of hemoglobin in-vivo, where its redox activity leads to peroxidative damage of membrane lipids and proteins. Spectrin, the major component of the red blood cell (RBC) membrane skeleton, is known to interact with hemoglobin and, here this interaction is shown to increase hemoglobin peroxidase activity in the presence of reducing substrate ABTS (2 0 , 2 0 -Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid). It is also shown that in the absence of reducing substrate, spectrin forms covalently cross-linked aggregates with hemoglobin which display no peroxidase activity. This may have implications in the clearance of ROS and limiting peroxidative damage. Spectrin is found to modulate the peroxidase activity of different hemoglobin variants like A, E, and S, and of isolated globin chains from each of these variants. This may be of importance in disease states like sickle cell disease and HbE-β-thalassemia, where increased oxidative damage and free globin subunits are present due to the defects inherent in the hemoglobin variants associated with these diseases. This hypothesis is corroborated by lipid peroxidation experiments. The modulatory role of spectrin is shown to extend to other heme proteins, namely catalase and cytochrome-c. Experiments with free heme and Raman spectroscopy of heme proteins in the presence of spectrin show that structural alterations occur in the heme moiety of the heme proteins on spectrin binding, which may be the structural basis of increased enzyme activity.

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Preparation and Properties of α- and β-Chains from Human Hemoglobin

Cited by 360 publications

References 15 publications

Multiple Geminate Ligand Recombinations in Human Hemoglobin

Multiple Geminate Ligand Recombinations in Human Hemoglobin

Electron capture by oxyhaemoglobin: an e. s. r. study

Erythroid spectrin binding modulates peroxidase and catalase activity of heme proteins

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