Calhoun Db

Biochemistry without oxygen

Published procedures for experimentation under anoxic conditions generally involve specialized apparatus that hinders the easy manipulation of experimental samples. We describe here some procedures that rapidly remove oxygen from experimental solutions, maintain anoxia with simple equipment for long periods of time, and do not interfere with normal sample addition and removal, spectrometric measurements, chromatographic manipulations, and the like. Anoxia can be achieved and maintained by the use of an enzyme system (glucose oxidase, glucose, catalase), or an inorganic oxygen-reducing system (ferrous pyrophosphate), or dithionite. Physical isolation of experimental samples from atmospheric oxygen can be maintained by continuous flushing with treated argon gas and/or by an overlay of heavy mineral oil.

Measurement and calibration of peptide group hydrogen-deuterium exchange by ultraviolet spectrophotometry.

Abstract An exceedingly simple and convenient method is described for measuring the hydrogen-deuterium exchange behavior of peptide bond-containing molecules by ultraviolet spectrophotometry. The exchange reaction is initiated by diluting a sample from H 2 O into D 2 O, or the reverse, and can be followed by an easily observable optical density change in the region of peptide absorbance. The method, unlike infrared and magnetic resonance approaches, requires only small amounts of material and, unlike the tritium-Sephadex method, is not restricted to the study of large molecules. Calibrations are provided for exchange rate as a function of pD and temperature and for the change in absorbance per mole peptide group. With this information, the exchange curve to be expected for any peptide group exposed to solvent can be predicted. Comparison with the measured data can then identify peptide-group hydrogen bonding and can also give a measure of the stability of the hydrogen-bonded structure.

Individual breathing reactions measured in hemoglobin by hydrogen exchange methods.

Protein hydrogen exchange is generally believed to register some aspects of internal protein dynamics, but the kind of motion at work is not clear. Experiments are being done to identify the determinants of protein hydrogen exchange and to distinguish between local unfolding and accessibility-penetration mechanisms. Results with small molecules, polynucleotides, and proteins demonstrate that solvent accessibility is by no means sufficient for fast exchange. H-exchange slowing is quite generally connected with intramolecular H-bonding, and the exchange process depends pivotally on transient H-bond cleavage. At least in alpha-helical structures, the cooperative aspect of H-bond cleavage must be expressed in local unfolding reactions. Results obtained by use of a difference hydrogen exchange method appear to provide a direct measurement of transient, cooperative, local unfolding reactions in hemoglobin. The reality of these supposed coherent breathing units is being tested by using the difference H-exchange approach to tritium label the units one at a time and then attempting to locate the tritium by fragmenting the protein, separating the fragments, and testing them for label. Early results demonstrate the feasibility of this approach.

Long-Range Electron Exchange Reactions of Excited Triplet Tryptophan in Proteins

Abstract Ten proteins that exhibit long-lived phosphorescence lifetimes at room temperature were examined for sensitivity to quenching by molecules that are external to the protein. The bimolecular quenching rate constant was found to decrease exponentially with the distance of the tryptophan from the protein surface. Theoretical analysis shows that this behavior is expected for an electron-exchange reaction between the buried tryptophan and quenchers in solution in the rapid diffusion limit. The results allow evaluation of the distance parameter, ρ, for electron transfer through the general protein matrix at 1.0 A, For a unimolecular donor-acceptor pair with ket = ko exp(-r/ρ), ko = 109sec−1.

Measurement of the haptoglobin concentration in plasma and other fluids by a simple spectrometric procedure.

A method for determining the concentration of available haptoglobin in plasma and other solutions is described. The procedure involves absorbance measurements in a mixture of plasma and hemoglobin before and after the addition of sodium dithionite. The haptoglobin content of the solution is calculated from the ratio of the absorbances at the Soret peaks of the oxy- and deoxyhemoglobin. The assay can be performed in minutes, and the error of measurement is well under 10%.