Chelates in the Study of Protein Interactions

Dordick, 1991). In addition, Fancy et al. (1996) as well as Fancy and Kodadek (1997, 1998) have applied the oxidation of tyrosine to form dityrosine to the study of protein-protein interactions using nickel-chelated 6 X His tagged fusion proteins in oxidative environments. 

The interaction between bisphosphonates and plasma proteins is not well understood, but it is known to be influenced by both calcium and iron ions [38]. The addition of calcium to human plasma in an in vitro study increased the proportion of plasma protein-bound versus free pamidronate, whereas the addition of calcium chelators reduced the proportion of drug bound. Similarly, the addition of ferric ions to plasma increased the proportion of plasma protein-bound versus free pamidronate [38]. 

Metal-enzyme complexes, a subgroup of metal-protein complexes, exhibit enzymatic activity consequent to readily dissociable combination with a variety of metal ions. Many of these studies have been performed with unpurified enzymes, and, even when pure enzymes were used, the stoichiometry of the interaction of the metal and enzyme has not been measured. Enhancement of enzymatic activity as a result of the addition of metal ions and its partial loss on their removal has been the chief criterion of assessment of physiological significance. Only in a few instances, e.g., enolase, has the stability and stoichiometry been studied in relation to function (Malmstrom, 1953, 1954). The study of metal complexes and particularly metal chelates (Bjerrum, 1941 Martell and Calvin, 1952 Calvin, 1954) has provided both new experimental and new theoretical backgrounds for the study of metals in relation to the specificity of enzyme action, metal-enzyme (Calvin, 1954), metal-substrate (Najjar, 1951), and metalloenzyme interaction, as well as metal-enzyme inhibition (James, 1953). 

Albumin is one of the most abundant proteins in plasma ( 0.63 mM) and it is reasonable to assume that any injected metallodrug will present certain interactions with this macromolecule, which could largely determine its bioavailability and toxicology [42]. A comprehensive study of the reactions of the N-labeled cisplatin with intact and chemically modified recombinant human albumin (rHA) as well as human serum album (HSA) has been performed by ID H and [ H, Nj 2D HSQC NMR spectroscopy [53]. Recombinant albumin is similar to serum albumin but has a higher thiol content ( 0.9 -SH per rHA and only 0.4-SH per HSA) and is structurally more homogeneous. NMR studies showed that the major sulfur-containing binding site involves Met in the form of an S, N chelate but not Cys-34, which is commonly believed to be the major platination site of HSA (Scheme 5.4) [73]. 

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