This proposal is concerned with a study of: the kinetics and mechanisms of the binding of oxygen and CO to various hemoglobins, the assembly reactions of Hb, and the functional properties of intermediates in the assembly process. In the assembly reactions, we are interested in determining at what stages and to what extent co-operativity in ligand binding appears. These reactions are followed in laser light-scattering, fluorescence anisotropy stopped-flow, tandem flow, and flow-flash experiments. From the light-scattering and fluorescence measurements, relative populations of intermediates can be determined. Tandem flow studies allow us to trap the transient intermediates and determine their functional properties. Detailed stopped-flow studies of ligation are directed toward unravelling the early phases of ligation and conformational changes in T-state human hemoglobin. Laser photolysis will probe details of geminal and bimolecular CO recombination in hemo-globins which are at the two extremes of known CO reactivity. In one hemoglobin, n-sec studies can be carried out on both the T and R states. These studies will provide information on energy barriers for ligation on the heme cavity and for protein channels which may be involved in the bimolecular reactions. Recent synthetic advances allow us to propose experiments directed toward elucidating the roles of the alpha and beta chains in the Root effect, the most extreme of Bohr effects. This is to be accomplished by synthesizing both the modified hemoglobin and various blocked or valency-hybrids, permitting ligation to the single reactive chain to be followed in both R and T states. The simplest of co-operative Hbs, one that is dimeric, without a Bohr effect or detectable alpha-beta heterogeneity, will be studied in an attempt to provide a description of the most elementary co-operative unit. In general, these studies are all directed toward improving and enlarging our understanding of protein-protein and protein-ligand interactions; in particular, the study is of subunit interactions and the binding of oxygen and Co to diverse hemoglobins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK036288-15
Application #
3234614
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1985-12-15
Project End
1990-11-30
Budget Start
1986-12-01
Budget End
1987-11-30
Support Year
15
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Nebraska Lincoln
Department
Type
Schools of Arts and Sciences
DUNS #
555456995
City
Lincoln
State
NE
Country
United States
Zip Code
68588
Parkhurst, K M; Brenowitz, M; Parkhurst, L J (1996) Simultaneous binding and bending of promoter DNA by the TATA binding protein: real time kinetic measurements. Biochemistry 35:7459-65
Parkhurst, K M; Parkhurst, L J (1995) Kinetic studies by fluorescence resonance energy transfer employing a double-labeled oligonucleotide: hybridization to the oligonucleotide complement and to single-stranded DNA. Biochemistry 34:285-92
Parkhurst, K M; Parkhurst, L J (1995) Donor-acceptor distance distributions in a double-labeled fluorescent oligonucleotide both as a single strand and in duplexes. Biochemistry 34:293-300
Parkhurst, K M; Hileman, R E; Saha, D et al. (1994) Thermodynamic characterization of the cooperativity of 40S complex formation during the initiation of eukaryotic protein synthesis. Biochemistry 33:15168-77
Parkhurst, L J; Larsen, T M; Lee, H Y (1994) Effects of wavelength on fitting Adair constants for binding of oxygen to human hemoglobin. Methods Enzymol 232:606-32
Hileman, R E; Parkhurst, K M; Gupta, N K et al. (1994) Synthesis and characterization of conjugates formed between periodate-oxidized ribonucleotides and amine-containing fluorophores. Bioconjug Chem 5:436-44
Mueser, T C; Parkhurst, L J (1993) Synthesis of dansyl ribonucleotides and their use in steady-state fluorescence anisotropy studies of nucleotide binding by initiation factor-2 (eIF-2) and histone H1. Int J Biochem 25:1689-96
Astatke, M; Parkhurst, L J (1993) A pulsed photolysis procedure for determining oxygen equilibrium parameters of low-affinity noncooperative hemoglobins. Photochem Photobiol 57:964-71
Astatke, M; McGee, W A; Parkhurst, L J (1992) A flow procedure to determine oxygen binding isotherms for low affinity and easily oxidized hemoglobins. Comp Biochem Physiol B 101:683-8
Parkhurst, K M; Parkhurst, L J (1992) Rapid preparation of native alpha and beta chains of human hemoglobin. Int J Biochem 24:993-8

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