Published In
bioRxiv
Document Type
Pre-Print
Publication Date
11-2023
Subjects
DNA -- Denaturation, DNA microarrays, Nucleic acid hybridization
Abstract
It was recently reported for two globular proteins and a short DNA hairpin in NaCl buffer that values of the transition heat capacities, Cp,DNA and Cp,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 Tm. Sequences of two DNA hairpins were designed to confer a near 20°C difference in their Tm’s. For the molecules, in NaCl and CsCl buffer the evaluated Cp,PRO and Cp,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.
Rights
Copyright (c) 2023 The Authors
Locate the Document
DOI
10.1101/2023.09.25.559360
Persistent Identifier
https://archives.pdx.edu/ds/psu/40977
Citation Details
Publshed as: Eskew, M. W., Reardon, P. W., & Benight, A. S. (2023). Calorimetric analysis using DNA thermal stability to determine protein concentration. bioRxiv, 2023-09.
Description
Pre-print version.
Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.