A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease^*

Abstract

A new method for measuring the hydrogen exchange of macromolecules in solution is described. The method uses tritium to trace the movement of hydrogen, and utilizes Sephadex columns to effect, in about 2 minutes, a separation between tritiated macromolecule and tritiated solvent great enough to allow the measurement of bound tritium. High sensitivity and freedom from artifact is demonstrated and the possible value of the technique for investigation of other kinds of colloidsmall molecule interaction is indicated. Competition experiments involving tritium, hydrogen, and deuterium indicate the absence of any equilibrium isotope effect in the ribonuclease-hydrogen isotope system, though a secondary kinetic isotope effect is apparent when ribonuclease is largely deuterated. Ribonuclease shows four clearly distinguishable kinetic classes of exchangeable hydrogens. Evidence is marshaled to suggest the independently measurable classes II, III, and IV (in order of decreasing rate of exchange) to represent "random-chain" peptides, peptides involved in α-helix, and otherwise shielded side-chain and peptide hydrogens, respectively.Protons attached to nitrogen, oxygen, and sulfur of molecules dispersed in liquid solution are, in general, able to exchange with mobile protons in the solvent. The rate of exchange of protons occupying specific molecular sites may be modified, or even largely controlled, by the details of molecular architecture in their immediate vicinity. Thus measurement of the kinetics of the hydrogen exchange of macromolecules can provide a fine probe for identifying and quantitating details of conformation and of changes in conformation of macromolecules.A practicable method for the measurement of hydrogen-exchange kinetics was first developed and used in the study of structure in proteins and polypeptides by Linderstrøm-Lang and his co-workers . Typically, in this method, deuterated protein is separated from deuterated solvent by freeze-drying, the protein is redissolved in water, and the kinetics of the loss of deuterium by the protein is followed by analyzing samples of solvent, after various times of protein incubation, for deuterium content. Deuterium content is determined by density measurement of tiny samples of solvent isolated from the protein by a second freeze-drying step. The method, though ingenious in conception, seems unusually demanding in execution and makes use of procedures which might be considered questionable. While the method is capable of considerable internal precision, a number of the operations involved, freezing, drying, heating (60°), redissolution,

Hydrogen exchange and mass spectrometry: A historical perspective

“…These advances, together with the development and commercialization of gel-filtration media, led to the accurate artifact-free tritium-gel-filtration HX technique [19], which dominated HX work for the next 20 years. A range of HX studies on proteins, nucleic acids, oligosaccharides, their synthetic analogs, and their multimolecular complexes followed.…”

From “Simple” DNA-Protein Interactions to the Macromolecular Machines of Gene Expression

“…These thermodynamic results led to kinetic questions and efforts to establish the rates at which dsDNA molecules breathe (open and close) locally. The hydrogen-tritium exchange method, initially developed by Englander (15) to study the opening and closing of protein domains, was used to examine the rates of dsDNA breathing of sequences of varying composition at temperatures below T m (45,58). These experiments showed that base pairs do open and close spontaneously within dsDNA, presumably also transiently exposing potential ssDNA templating sequences to be trapped by polymerases or related helper proteins in the initiation of DNA replication and transcription.…”

Conformational Studies of Oxytocin, Lysine Vasopressin, Arginine Vasopressin, and Arginine Vasotocin by Carbon-13 Nuclear Magnetic Resonance Spectroscopy

“…Nevertheless, the conformational analysis involves several approximations: (1) the relationship between the dihedral angle 0 (NH-CaH) and the measured three-bond coupling constant 3JHNCH (8,9)-a spectral parameter most useful when considered in combination with potential energy maps (10,11); (2) temperature (12) and solvent (13,14) dependences of chemical shifts and proton exchange rates (15)(16)(17) of peptide hydrogens as related to their solvent-exposed or solvent-shielded state.…”