Reactions of cisplatin (cis-[

Human serum albumin prepared by blood fractionation for clinical purposes was found to degrade when stored at or above 30°C. Mass spectrometry and N-terminal sequencing of the protein identified degradation corresponding to the loss of the first two residues, aspartic acid and alanine. The reaction was shown to be dependent upon temperature and the N-terminal a-amino group. In addition, comparison with serum albumins derived from other species showed that the instability of the N-terminus was specific to the human albumin sequence. An intact aspartyl-alanyl dipeptide, purified from degraded albumin solutio'ns, differed substantially from a synthetic dipeptide on amino acid analysis, N-terminal sequencing and NIMR. It is suggested that the released dipeptide may be cyclic, implying a novel cleavage mechanism.

Forty-eight strains of Thermus isolated from hot springs in Yellowstone National Park, Wyoming, USA, and eight reference strains were subjected to a numerical taxonomic analysis using Gower's coefficient (SG) with single and average linkage clustering. Two major groups were distinguished, which could be differentiated by colony morphology, ability to reduce nitrate and proteolytic activity. Cluster 1 contained Thermus aquaticus YT-1, the type strain of the species, and cluster 2 contained authentic strains of 'T.flavus' and 'T. therrnophilus'. T. ruber was recovered as a single member cluster. The mol % G + C of DNA from representative strains from each cluster was 64.4 to 66.8 for cluster 1, 62.2 to 67.1 for cluster 2 and 62.5 for T. ruber.

Recombinant human HbA, produced by co-expressing a-globin and P-globin chains in the yeast Saccharomyces cerevisiae, has been characterised extensively both physically and functionally. Structural studies using N-terminal sequence analysis, peptide mapping, amino acid composition analysis and electrospray MS demonstrated that the recombinant protein was identical to standard HbA purified from erythrocytes. The functional properties of the recombinant protein were assessed using equilibrium and kinetic measurements of oxygen and carbon monoxide binding. The oxygen-binding studies demonstrated that the yeast-derived HbA behaved as a fully functional, cooperative tetramer (Hill coefficient, 2.9), exhibited a normal Bohr effect and response to phosphate, and displayed a rate of oxygen dissociation identical to that of the native human molecule. The recombinant protein also showed the same characteristics of carbon monoxide combination as the standard protein. These studies demonstrate that yeast provides an ideal system for the production of Hb for structural and functional analysis and a potentially useful source of HbA for formulation into a Hb-based oxygen carrier.For many years there has been interest in the potential of cell-free haemoglobin as a substitute for red cells in oxygen delivery (for reviews see Winslow, 1989;Vandegriff and Winslow, 1991). Research into the development of red-cell substitutes has intensified because of increasing concern over viral contamination of donated blood. To be clinically useful, cell-free Hb requires modification, both to stabilise the tetramer against dissociation into dimers and to reduce the oxygen affinity, thereby allowing efficient tissue oxygenation. Such properties have been achieved by cross-linking Hb chemically using a variety of intramolecular and intermolecular cross-linking agents (for reviews see Chang, 1988;Vandegriff and Winslow, 1991).Currently, Hb for modification is purified from outdated stocks of donated blood. Increasingly, however, it appears that this source of Hb will become limiting (Surgenor et al., 1990). Recombinant-DNA techniques now make it possible to produce recombinant Hb (rHb) in genetically engineered microorganisms. This ensures a source of protein which is free of human-blood-derived infectious agents such as hepatitis B and human immunodeficiency virus.Human rHb was first synthesised using Escherichia coli as a host (Nagai and Therrgersen, 1984;Hoffman and Nagai, 1991 as insoluble, intracellular fusion proteins which required solubilisation followed by specific proteolytic cleavage to produce authentic N-termini. Functional tetrameric Hb was then reconstituted in vitro by mixing the globin subunits and exogenous haem. While this approach has proved valuable for production of small quantities of wild-type HbA and modified Hb for structural and functional studies, it would be extremely costly to synthesise rHb on a large scale using these methods.More recently, rHb has been synthesised in E. coli (Hoffman et al., 1990) and Saccharomyces...

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