Spectroscopic and functional characterization of T state hemoglobin conformations encapsulated in silica gels

Uri Samuni, David Dantsker, Laura J. Juszczak, Stefano Bettati, Luca Ronda, Andrea Mozzarelli, Joel M. Friedman

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

Oxygen binding curves of sol-gel-encapsulated deoxy human adult hemoglobin (HbA) have previously revealed two distinct noncooperative populations with oxygen binding affinities approximately 1000 and 100 times lower than that of the high-affinity R state. The two populations which have been termed the low-affinity (LA) and high-affinity (HA) T states can be selectively stabilized using two different encapsulation protocols for deoxy-HbA. The present study seeks to understand the factors giving rise to these different affinity states. Visible and UV resonance Raman spectroscopies are used to characterize the conformational properties of both the deoxy and deoxy-turned-carbonmonoxy (CO) derivatives of HbA derived from the two encapsulation protocols. The geminate and bimolecular recombination of CO to the photodissociated CO derivatives is used to characterize the functional properties of the slowly evolving encapsulated populations. The results show that the initial deoxy-HbA populations are conformationally indistinguishable with respect to encapsulation protocol. The addition of CO to sol-gel-encapsulated deoxy-HbA triggers a detectable progression of conformational and functional changes. Visible resonance Raman spectra of the CO photoproduct reveal a progression of changes of the iron-proximal histidine stretching frequencies: 215, 222, 227, and 230 cm-1. The low and high values correspond to the initial deoxy T state and liganded R (R2) state species, respectively. The 222 and 227 cm-1 species are generated using encapsulation protocols that give rise to what are termed the LA and HA T states, respectively. The UV resonance Raman spectra of these and related species indicate that the progression from deoxy T to LA to HA is associated with a progressive loosening of T state constraints within the hinge and switch regions of the α1β 2 interface. The time scale for the progression is determined by a balance between the ligation-initiated evolution toward high-affinity conformations and factors such as allosteric effectors, gel matrix, and added glycerol that slow ligand-binding-induced relaxation. Thus, it appears that the encapsulation protocol-dependent rate of ligand-binding-induced relaxation determines the functional properties of the initially encapsulated deoxy-HbA population.

Original languageEnglish (US)
Pages (from-to)13674-13682
Number of pages9
JournalBiochemistry
Volume43
Issue number43
DOIs
StatePublished - Nov 2 2004

Fingerprint

Silica Gel
Encapsulation
Conformations
Population
Gels
Polymethyl Methacrylate
Sol-gels
Raman scattering
Oxygen
Ligands
Derivatives
Raman Spectrum Analysis
Hinges
Histidine
Glycerol
Genetic Recombination
Stretching
Ligation
Raman spectroscopy
Hemoglobins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Spectroscopic and functional characterization of T state hemoglobin conformations encapsulated in silica gels. / Samuni, Uri; Dantsker, David; Juszczak, Laura J.; Bettati, Stefano; Ronda, Luca; Mozzarelli, Andrea; Friedman, Joel M.

In: Biochemistry, Vol. 43, No. 43, 02.11.2004, p. 13674-13682.

Research output: Contribution to journalArticle

Samuni, U, Dantsker, D, Juszczak, LJ, Bettati, S, Ronda, L, Mozzarelli, A & Friedman, JM 2004, 'Spectroscopic and functional characterization of T state hemoglobin conformations encapsulated in silica gels', Biochemistry, vol. 43, no. 43, pp. 13674-13682. https://doi.org/10.1021/bi048531d
Samuni U, Dantsker D, Juszczak LJ, Bettati S, Ronda L, Mozzarelli A et al. Spectroscopic and functional characterization of T state hemoglobin conformations encapsulated in silica gels. Biochemistry. 2004 Nov 2;43(43):13674-13682. https://doi.org/10.1021/bi048531d
Samuni, Uri ; Dantsker, David ; Juszczak, Laura J. ; Bettati, Stefano ; Ronda, Luca ; Mozzarelli, Andrea ; Friedman, Joel M. / Spectroscopic and functional characterization of T state hemoglobin conformations encapsulated in silica gels. In: Biochemistry. 2004 ; Vol. 43, No. 43. pp. 13674-13682.
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AU - Samuni, Uri

AU - Dantsker, David

AU - Juszczak, Laura J.

AU - Bettati, Stefano

AU - Ronda, Luca

AU - Mozzarelli, Andrea

AU - Friedman, Joel M.

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N2 - Oxygen binding curves of sol-gel-encapsulated deoxy human adult hemoglobin (HbA) have previously revealed two distinct noncooperative populations with oxygen binding affinities approximately 1000 and 100 times lower than that of the high-affinity R state. The two populations which have been termed the low-affinity (LA) and high-affinity (HA) T states can be selectively stabilized using two different encapsulation protocols for deoxy-HbA. The present study seeks to understand the factors giving rise to these different affinity states. Visible and UV resonance Raman spectroscopies are used to characterize the conformational properties of both the deoxy and deoxy-turned-carbonmonoxy (CO) derivatives of HbA derived from the two encapsulation protocols. The geminate and bimolecular recombination of CO to the photodissociated CO derivatives is used to characterize the functional properties of the slowly evolving encapsulated populations. The results show that the initial deoxy-HbA populations are conformationally indistinguishable with respect to encapsulation protocol. The addition of CO to sol-gel-encapsulated deoxy-HbA triggers a detectable progression of conformational and functional changes. Visible resonance Raman spectra of the CO photoproduct reveal a progression of changes of the iron-proximal histidine stretching frequencies: 215, 222, 227, and 230 cm-1. The low and high values correspond to the initial deoxy T state and liganded R (R2) state species, respectively. The 222 and 227 cm-1 species are generated using encapsulation protocols that give rise to what are termed the LA and HA T states, respectively. The UV resonance Raman spectra of these and related species indicate that the progression from deoxy T to LA to HA is associated with a progressive loosening of T state constraints within the hinge and switch regions of the α1β 2 interface. The time scale for the progression is determined by a balance between the ligation-initiated evolution toward high-affinity conformations and factors such as allosteric effectors, gel matrix, and added glycerol that slow ligand-binding-induced relaxation. Thus, it appears that the encapsulation protocol-dependent rate of ligand-binding-induced relaxation determines the functional properties of the initially encapsulated deoxy-HbA population.

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