# DNA-based assay for calorimetric determination of protein concentrations in pure or mixed solutions

## TL;DR

A new DNA-based calorimetric method is developed to accurately determine protein concentrations in pure or mixed solutions.

## Contribution

A novel calorimetric method using DNA hairpins to measure protein concentrations and infer molecular stoichiometry is introduced.

## Key findings

- Transition heat capacities of proteins and DNA hairpins are equivalent at equal concentrations.
- The DNA-based calorimetric method accurately determines protein concentrations and reveals molecular stoichiometry.
- The method works across different DNA sequences, buffer salts, and transition temperatures.

## Abstract

It was recently reported that values of the transition heat capacities, as measured by differential scanning calorimetry, for two globular proteins and a short DNA hairpin in NaCl buffer are essentially equivalent, at equal concentrations (mg/mL). To validate the broad applicability of this phenomenon, additional evidence for this equivalence is presented that reveals it does not depend on DNA sequence, buffer salt, or transition temperature (Tm). 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. Sequences of two DNA hairpins were designed to confer a near 20°C difference in their Tm values. In all cases, evaluated protein concentrations determined from the DNA standard curves agreed with the UV-Vis concentration for monomeric proteins. For multimeric proteins evaluated concentrations were greater than determined by UV-Vis suggesting the calorimetric approach can also be an indicator of molecular stoichiometry.