Hydrogen/deuterium exchange on protein solutions containing nucleic acids: utility of protamine sulfate.
Obtaining global hydrogen/deuterium (H/D) exchange data on proteins is an important first step in amide proton exchange experiments. Important information such as the mode of exchange, the cooperativity of folding/unfolding reactions, and the effects of ligand binding can be readily obtained in global exchange experiments. Many interesting biological systems are complexes containing both proteins and nucleic acids. The low pH conditions required to quench H/D exchange reactions result in the formation of stable protein/nucleic acid precipitates which interfere with the liquid chromatography step of the experiment and preclude obtaining mass spectrometric data. In this work we show that the precipitation of proteins and nucleic acids is electrostatic in nature and can be prevented by high ionic strength and by removing nucleic acids by protamine sulfate. Using protamine sulfate in quenching solution, we were able to obtain global H/D data with protein samples containing large amounts of DNA or RNA.
Poliakov
Anton
A
Department of Microbiology, University of Alabama at Birmingham, 1900 University Boulevard, Birmingham, AL 35294, USA. poliakov@uab.edu
Jardine
Paul
P
Prevelige
Peter E
PE
D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry*; D041961:Deuterium Exchange Measurement / Q000379:methods; D011479:Protamines / Q000737:chemistry*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
D011479:Protamines; D011506:Proteins; D012313:RNA; D004247:DNA; D003903:Deuterium