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 (R(2)) 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 alpha(1)beta(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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Samuni U,Dantsker D,Juszczak LJ,Bettati S,Ronda L,Mozzarelli A,Friedman JMdoi
10.1021/bi048531dsubject
Has Abstractpub_date
2004-11-02 00:00:00pages
13674-82issue
43eissn
0006-2960issn
1520-4995journal_volume
43pub_type
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