Calorimetric analysis using DNA thermal stability to determine protein concentration

It was recently reported for two globular proteins and a short DNA hairpin in NaCl buffer that values of the transition heat capacities, C_p,DNA and C_p,PRO, for equal concentrations (mg/mL) of DNA and proteins, are essentially equivalent (differ by less than 1%). Additional evidence for this equivalence is presented that reveals this phenomenon does not depend on DNA sequence, buffer salt, or T_m. Sequences of two DNA hairpins were designed to confer a near 20°C difference in their T_m's. For the molecules, in NaCl and CsCl buffer the evaluated C_p,PRO and C_p,DNA were equivalent. Based on the equivalence of transition heat capacities, a calorimetric method was devised to determine protein concentrations in pure and complex solutions. The scheme uses direct comparisons between the thermodynamic stability of a short DNA hairpin standard of known concentration, and thermodynamic stability of protein solutions of unknown concentrations. In all cases, evaluated protein concentrations determined from the DNA standard curve agreed with the UV-Vis concentration for monomeric proteins. For samples of multimeric proteins, streptavidin (tetramer), Herpes Simplex Virus glycoprotein D (trimer/dimer), and a 16 base pair DNA duplex (dimer), evaluated concentrations were greater than determined by UV-Vis by factors of 3.94, 2.65, and 2.15, respectively.