Resonances for lactate are broadened in 500 MHz 1H NMR spectra of human blood plasma and only about one‐third is visible in Hahn spin‐echo spectra. Similar effects are observed for some other carboxylate anions. Lactate added to the high‐M r fraction of plasma can give rise to peaks which are too broad to observe in either single‐pulse or spin‐echo spectra. Addition of agents such as NH4Cl of SDS dramatically increases the intensities of lactate peaks. Some glycoproteins appear to broaden lactate resonances.

(1) The binding of the synergistic anion oxalate and Ga3+ to human serum transferrin (HTF, 80 kDa) and its recombinant N-lobe (HTF/2N, 40 kDa) has been studied by one- and two-dimensional 1H NMR spectroscopy, at 310 K, pH*7.25. (2) Specific protein resonances are sensitive to oxalate binding (fast exchange on the NMR time scale) and allowed determination of the apparent binding constant for oxalate binding to the N-lobe (log K 4.04). (3) Slow exchange between apo-HTF and Ga-loaded HTF or HTF/2N was observed. Binding of Ga3+ appeared to be accompanied by small changes in the orientations of residues in hydrophobic pockets in the interdomain hinge region close to the metal binding site. (4) Under the conditions used, preferential binding of Ga3+ (added as Ga(NTA)2) to the C-lobe of HTF was observed. Binding to the C-lobe markedly perturbed resonances in the glycan N-acetyl region of the spectrum, suggesting that metal binding is communicated to the surface of the protein. This could be important in receptor recognition of metallotransferrins. (5) The displacement of Ga3+ from Ga-ox-HTF with Fe3+ was studied, and the paramagnetic broadening effects allowed identification of resonances from groups close to Fe3+. The passage of Fe3+ from the exterior to the interior of the protein was followed by 1H NMR spectroscopy, and the half-life for Ga(3+)-Fe3+ exchange was determined to be 4.3 h (310 K).

We have studied the binding of Al3+ to human serum apotransferrin (80 kDa) and recombinant N-lobe human apotransferrin (40 kDa) in 0.1 M-sodium bicarbonate solution at a pH meter reading in 2H2O (pH*) of 8.8 using 1H n.m.r. spectroscopy. The results show that for the intact protein, preferential binding of Al3+ to the N-lobe occurs. Molecular modelling combined with an analysis of ring-current-induced shifts suggest that n.m.r. spectroscopy can be used to probe hinge bending processes which accompany metal uptake in solution.

Reactions of Al3+ (50-500 mumol l-1) with intact blood plasma and its low relative molecular mass ultrafiltrate (< 5 kDa) have been studied by proton nuclear magnetic resonance spectroscopy. Binding to citrate was detected and was reversed by addition of desferrioxamine. The use of combined exponential and sine-bell functions for the resolution enhancement of spectra of plasma is illustrated.

Conjugation reactions of glutathione (GSH) and related thiols with diethyl maleate (DEM) and other alpha, beta-unsaturated carbonyl compounds have been investigated by 1H NMR spectroscopy. The products from the reaction with DEM and diethyl fumarate (DEF) are shown to be the diastereomers of S-(alpha,beta-diethoxycarbonylethyl)glutathione. During the course of the reaction, DEM isomerized to DEF, and the rate of isomerization was dependent upon whether the solvent was 1H2O or 2H2O. The observed rate data exhibit apparent second order kinetic behavior. The reaction of maleate with GSH was considerably slower, and solvent-dependent isomerization was observed, while little reaction of fumarate with GSH was observed at pH 6.5. Reaction of DEM with N-acetyl-L-cysteine followed a similar course to that of GSH, and although L-cysteine reacted rapidly with DEM, it did not promote the isomerization of DEM. Reactions involving penicillamine and N-acetylpenicillamine were considerably slower. Conjugation reactions catalyzed by commercial GSH transferases and selected rat and human purified isoenzymes were also investigated. Of those isoenzymes studied, rat GSH transferase 4-4 was found to exert the greatest degree of stereo control in conjugation reactions with DEF.