Determination of protein--ligand equilibria by difference spectroscopy. Hemerythrin--ligand thermodynamic studies.
A difference spectrophotometric method for the rapid determination of equilibrium constants for protein--ligand interactions has been developed. The method requires no knowledge of the extinction coefficient of either reactants or products. Furthermore the method allows rapid determination of the temperature dependence of a reaction and thus leads to rapid determination of thermodynamic parameters. The method has been tested by following the interactions of ligands with hemerythrin, the nonheme iron, oxygen storage protein isolated from Phasocolopsis gouldii. The reactions were studied at various temperatures and ionic strengths, and standard thermodynamic parameters were determined. The standard thermodynamic parameters for the conversion of metaquohemerythrin to methydroxyhemerythrin were found to be delta H degrees = 5.8 +/- 1.3 kcal mol-1 and delta S degrees = -11.5 +/- 1.5 cal mol-1 deg-1. For the reaction of metaquohemerythrin with thiocyanate ion to produce metthiocyanatohemerythrin delta H degrees = --13.0 +/- 1.6 kcal mol-1 and delta S degrees - --25.3 +/- 5.5 cal mol-1 deg-1. For the reaction of thiocyanate ion with methydroxy-hemerythrin delta H degrees = --6.6 +/- 0.8 kcal mol-1 and delta S degrees = --38.3 +/- 4.0 cal mol-1 deg-1. Perchlorate ion decreases the affinity of metaquohemerhythrin for thiocyanate ion. This is reflected in both the entropy and enthalpy being more unfavorable for the reaction in the presence of perchlorate ion.
D000818:Animals; D000845:Annelida; D006422:Hemerythrin; D007501:Iron; D008024:Ligands; D008433:Mathematics; D008667:Metalloproteins; D009994:Osmolar Concentration; D011506:Proteins; D013053:Spectrophotometry; D013816:Thermodynamics
D006422:Hemerythrin; D008024:Ligands; D008667:Metalloproteins; D011506:Proteins; D007501:Iron