Thermochemistry of the Avidin‐Biotin Reaction

Abstract

The enthalpy for the binding of biotin to avidin has been determined a t 25, 34 and 43 "C (pH 9, ammonium acetate buffer): AH (25 "C) = -22.5 f 0.1 kcal/mol biotin; AC,, = -237 &-12 cal x mol biotin-l x K-l.Solubilities and enthalpies of solution of biotin in water and in ethanol were determined at 25 "C. The transfer process for biotin between water and ethanol was considered as a simple model for the protein binding process.The properties of avidin and the nature of the avidin-biotin binding reaction have been studied extensively, in particular by Green and coworkers [l-51. It was shown that avidin has four identical and independent binding sites for biotin. The standard free energy of binding, AGO, was determined to be -20.5 kcal/mole biotin and from calorimetric measurements around 25 "C, the corresponding enthalpy change was found to be within uncertainty limits the same. AH was shown to be independent of pH (between 5 and 9) and independent of the buffer used [3].I n the present work, some of Green's calorimetric measurements have been repeated. Measurements were further extended to three well-defined temperatures in order to arrive a t a A C,, value for the process.Solubilities and calorimetric enthalpies of solution of biotin in water and in ethanol were determined. From the results of these measurements, AH and AS for the transfer of biotin between water and ethanol were calculated. This transfer process may be looked upon as a simple model for the studied biotin-avidin binding reaction. EXPERIMENTAL PROCEDURE MaterialsAvidin was purchased from Worthington (Freehold, NewJersey, U.S.A.) and was used without further purification (lot No. Av IAA; 11.6 unitslg). Avidin solutions were freshly prepared for each experiment.Biotin (puriss grade, BDH) was further purified by recrystallization from water. The crystals were Presented in part at First European Biophysics Congress, Baden (Wien) 1971. dried in air a t I10 "C for 12 h after which they were vacuum treated for 24 h a t room temperature. Alkaline titration gave an equivalent weight of 244.8 6 0.2 (99.5°/0).Glass-distilled water was used in the preparation of the calorimetric solutions. Ammonium acetate, used as buffer substance in the calorimetric experiments, was of analytical grade (BDH). I n the solution experiments, 99.5O/, ethanol was used. No impurities could be detected by gas chromatography. Calorimetric Equipment and ProcedureFor the protein-binding experiments an LKB 10700-2 batch microcalorimeter was used. The instrument was fitted with reaction cells of 18-carat gold. The compartments in one of the cells were charged with 2 and 4 ml of avidin and biotin solution, respectively. The reference cell was charged with the same quantities of buffer solution. For each calorimetric run, both cells were treated in the same manner with respect to rinsing and charging procedures.Measurements were made at three temperatures, 25, 34 and 43 "C, respectively. The exact reaction temperatures were determined by means of a Calibrated thermistor placed inside t...

Chimeric avidin shows stability against harsh chemical conditions—biochemical analysis and 3D structure

“…The enthalpies of the avidin-biotin and chimeric avidin-biotin reactions at 258C were À107.6 and À112.6 kJ/ mol, respectively. The value for avidin is in line with the previously determined values of À94.2 (Suurkuusk and Wadso, 1972) and À98.0 kJ/mol (Swamy, 1995). The magnitude of the interaction enthalpy is dependent on bond lengths and bond angles.…”

Enthalpy-entropy compensation and heat capacity changes for protein-ligand interactions: general thermodynamic models and data for the binding of nucleotides to ribonuclease A

“…The partial specific heat capacity, C , , of globular proteins has been found to be about 0.32 cal/(g.deg) [e.g., for chymotrypsin (Suurkuusk, 1974)]. A large contribution to the C, must be due to vibrational and rotational contributions from the peptide bonds and residue side chains and from interactions between the protein and water (Kanehisa & Ik-number of intact intramolecular hydrogen bonds or van der Waals interactions.…”

“…The dependence of AC',(app) on the value ,K is shown. The data were simulated by using eq 4 and 8 and the parameters given in the legend of Figures 1 and 2. egami, 1977; Suurkuusk, 1974;Yang & Rupley, 1979). While it is true that the C, of a protein is, to a first approximation, equal to the sum of the heat capacities of the individual amino acids (Suurkuusk, 1974), it is also likely that a small portion of the Cp of a globular protein is due to the existence of various microscopic states which differ only slightly in terms of the…”

Molecular dynamics study of unbinding of the avidin-biotin complex

“…1) that functions as an activated CO2 carrier in some biochemical reactions (e.g., in the carboxylation of bind up to four molecules of biotin. The binding affinity is extremely high, characterized through a Helmholtz free energy and enthalpy change of about 20 kcal/mol (Suurkusk and Wadso, 1972;Swamy, 1995;Miyamoto and Kollman, 1993) and a binding constant of Kd = 10-15 (Green, 1975).…”