TY - JOUR
T1 - Temperature titration
T2 - A new approach to the thermodynamics of oxygen binding to hemoglobin
AU - Gryczynski, Zygmunt
AU - Gering, Henry
AU - Bucci, Enrico
N1 - Funding Information:
1This work was supported in part by PHS NIH Grant P01-HL-48517. Computer time and facilities were supported in part by the computer network of the University of Maryland at Baltimore, and at College Park, MD.
PY - 1998/1/15
Y1 - 1998/1/15
N2 - A cell was constructed in order to study hemoglobin's reaction with gaseous ligands. The temperature of the hemoglobin sample is systematically altered within a given temperature range (275-310°K), while the percentage of oxygen in the equilibrating gas is kept constant. The equilibration time of the sample at each temperature step depends on sample concentration, ligand affinity, and absolute temperature; in most cases, the equilibration time is on the order of minutes. The construction of the optical compartment allows the experimenter to vary the optical pathlength using specially designed spacers, thus making it possible to study hemoglobin-ligand interactions over a wide range of protein concentrations (0.1-200 mg/ml). Optical glass is used in the construction of the cuvette in order to optimize its optical stability over a long period of time. At equilibrium the absorption spectrum of the sample is collected and decomposed into the relative contributions of oxy-Hb, deoxy-Hb, and ferric-Hb, thus revealing the fraction of oxyhemoglobin as well as any baseline drifts and protein degradation. Temperature steps of 1°K are already sufficient to change the absorption spectra in a significant way. This type of setup is also advantageous in that the experimenter can change the sample at any point (temperature) without having to restart the entire experiment. This makes it possible to study the oxygen binding characteristics of unstable hemoglobins. Analyses of the binding curves obtained with this technique immediately yield the overall oxygen binding constants β(i) together with the respective standard enthalpies ΔH(i).
AB - A cell was constructed in order to study hemoglobin's reaction with gaseous ligands. The temperature of the hemoglobin sample is systematically altered within a given temperature range (275-310°K), while the percentage of oxygen in the equilibrating gas is kept constant. The equilibration time of the sample at each temperature step depends on sample concentration, ligand affinity, and absolute temperature; in most cases, the equilibration time is on the order of minutes. The construction of the optical compartment allows the experimenter to vary the optical pathlength using specially designed spacers, thus making it possible to study hemoglobin-ligand interactions over a wide range of protein concentrations (0.1-200 mg/ml). Optical glass is used in the construction of the cuvette in order to optimize its optical stability over a long period of time. At equilibrium the absorption spectrum of the sample is collected and decomposed into the relative contributions of oxy-Hb, deoxy-Hb, and ferric-Hb, thus revealing the fraction of oxyhemoglobin as well as any baseline drifts and protein degradation. Temperature steps of 1°K are already sufficient to change the absorption spectra in a significant way. This type of setup is also advantageous in that the experimenter can change the sample at any point (temperature) without having to restart the entire experiment. This makes it possible to study the oxygen binding characteristics of unstable hemoglobins. Analyses of the binding curves obtained with this technique immediately yield the overall oxygen binding constants β(i) together with the respective standard enthalpies ΔH(i).
UR - http://www.scopus.com/inward/record.url?scp=0032518562&partnerID=8YFLogxK
U2 - 10.1006/abio.1997.2483
DO - 10.1006/abio.1997.2483
M3 - Article
C2 - 9451501
AN - SCOPUS:0032518562
SN - 0003-2697
VL - 255
SP - 176
EP - 182
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -