Evaluation of total purchasing pilots in England and Scotland and implications for primary care groups in England: personal interviews and analysis of routine data

Objectives: To evaluate the reported achievements of the 52 first wave total purchasing pilot schemes in 1996-7 and the factors associated with these; and to consider the implications of these findings for the development of the proposed primary care groups.

Glycerol-induced development of catalytically active conformation of Crotalus adamanteus L-amino acid oxidase in vitro.

The reconstitutable apoprotein of Crotalus adamanteus L-amino acid oxidase was prepared using hydrophobic interaction chromatography. After reconstitution with flavin adenine dinucleotide, the resulting protein was inactive, with a perturbed conformation of the flavin binding site. Subsequently, a series of cosolvent-dependent compact intermediates was identified. The nearly complete activation of the reconstituted apoprotein and the restoration of its native flavin binding site was achieved in the presence of 50% glycerol. We provide evidence that in addition to a merely stabilizing effect of glycerol on native proteins, glycerol can also have a restorative effect on their compact equilibrium intermediates, and we suggest the hydrophobic effect as a dominating force in this in vitro-assisted restorative process.

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

We have inserted a fourth protein ligand into the zinc coordination polyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). The three-dimensional structures of threonine-199-->aspartate (T199D) and threonine-199-->glutamate (T199E) CAIIs, determined by x-ray crystallographic methods to resolutions of 2.35 Angstrum and 2.2 Angstrum, respectively, reveal a tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered carboxylate side chain, which displaces zinc-bound hydroxide. Although the stereochemistry of neither engineered carboxylate-zinc interaction is comparable to that found in naturally occurring protein zinc-binding sites, protein-zinc affinity is enhanced in T199E CAII demonstrating that ligand-metal separation is a significant determinant of carboxylate-zinc affinity. In contrast, the three-dimensional structure of threonine-199-->histidine (T199H) CAII, determined to 2.25-Angstrum resolution, indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an additional protein ligand represents a general approach for increasing protein-metal affinity if the side chain can adopt a reasonable conformation and achieve inner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity metal ion biosensors.