INFLUENCE OF TRANSITION RATES AND SCAN RATE ON KINETIC SIMULATIONS OF DIFFERENTIAL SCANNING CALORIMETRY PROFILES OF REVERSIBLE AND IRREVERSIBLE PROTEIN DENATURATION

The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states: [GRAPHICS] We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (C(p)) in terms of the reduced variables v/k(i) and k1/k3, where v is the scan rate. The three-state model reduces to the two-state reversible or irreversible models for very large or very small values of k1/k3, respectively. The apparent transition temperature (T(app)) is always reduced by the irreversible step (U --> D). For all values of k3, T(app) is independent of v/k1 at sufficiently slow scan rates, even when denaturation is highly irreversible, but increases identically for all models at fast scan rates in which case the excess specific heat profile is determined by the rate of unfolding. Accurate values of DELTAH and DELTAS can be obtained for the reversible step only when k1 is more than 2000-50 000 times greater than k3. In principle, approximate values for the ratio k1/k3 can be obtained from plots of fraction unfolded vs fraction irreversibly denatured as a function of temperature; however, the fraction irreversibly denatured is difficult to measure accurately by DSC alone. A guide to determining whether a reversible analysis is appropriate for analyzing DSC scans for apparently irreversible denaturation is described on the basis of these plots. While a high level of irreversibility can be tolerated at greater than 95% unfolding, at lower levels of unfolding only very low levels of irreversibility (1-14% at 75% unfolding and less than 2% at 50% unfolding) can occur for accurate determination of DELTAH and DELTAS and deconvolution of complex profiles using models that assume reversibility. Under conditions of higher irreversibility, a kinetic analysis is both more appropriate and more informative.