A spectroscopic study of nickel(II)-bovine serum albumin binding and reactivity.

The pH dependence of the uv/visible and CD spectra of the 1:1 Ni(BSA) complex in aqueous solutions is interpreted in terms of a major square-planar form and an octahedral form. At pH 7.4, the two forms, respectively, account for ca. 70% and 30% of the total Ni(II). The two forms are in rapid equilibrium with each other and so both probably involve Ni(II) binding to the N-terminal region of the albumin protein. The kinetics of the equilibrium reaction of Ni(BSA) with His were studied at 37 degrees C in buffered media of pH 7.4 and 9.3. In line with predictions, the two Ni(BSA) forms show markedly different reactivities, with the square-planar form being the more thermodynamically stable and the less reactive. The octahedral form reacts with an observed zero-order dependence on His concentration while the square-planar form shows both zero-order and first-order dependence, the latter being the more dominant. The significance of the slow equilibrium rate at pH 7.4 to the possible physiological role of Ni-albumin in blood serum is discussed.

Copper-albumin: what is its functional role?

The small copper fraction in animal blood that is bound to albumin protein is generally considered to have a transport role for the metal. However, several studies have concluded that copper ion incorporation into copper-enzymes requires caeruloplasmin to act as the transport form. The kinetic rates of Cu(albumin) reactions are also not in keeping with a general transport function. Only in the portal bloodstream does the Cu(albumin) appear to have a well-established transport role, i.e. in delivering the metal ions from the intestine to the liver. Thereafter the evidence as to its function is less certain; it could act as a storage form of the metal or have no role at all. Similar considerations apply to other metal-albumin fractions e.g. those of zinc and nickel.

Transport and storage of metals.

Current understanding of the means by which the trace metals copper, iron, and zinc are transported and stored in the human body is reviewed. Although metal-related inherited metabolic diseases manifest themselves as deficiencies in metalloenzymes, these deficiencies in fact arise from disturbances in the absorption/rejection or transport or storage phases of the metal metabolism. The considerable information available on iron metabolism shows these phases to be closely interrelated with a number of feedback mechanisms operating as controls on the various metabolic stages. Copper and zinc have vastly different roles in their enzymes but, following their close coordination chemistry properties, have similar metabolic routes. Both make use of albumin for transport and of metallothionein at the absorption and hepatic-binding stages. In contrast to iron, copper and zinc have no well-defined storage system; instead they make use of body reserves distributed in various tissues. Our knowledge of the transport and storage systems of the trace metals is still very incomplete. More research is needed on the dynamics of movement of the trace metals between the various body tissues and fluids. Further advances will also come from the study of related metal inherited diseases in other animal species.

The copper-molybdenum antagonism in ruminants. I. The formation of thiomolybdates in animal rumen.

The formation of thiomolybdates, MoOxS4--x2--(x = 0, 1, 2, or 3), from molybdate and sulphide salts in aqueous media has been studied under conditions which simulate the fluid phase in the rumen. The influences of the sulphide:molybdenum ratio, pH and phosphate levels on the nature of the species formed were investigated. The thiomolybdates, in particular the MoS42-- ion, have been implicated as the active intermediates in the widespread molybdenum induced copper deficiency that affects ruminants. The results presented here show that, under physiological conditions, di- and trithiomolybdates will form more readily than tetrathiomolybdate.

The formation and nature of the mixed valence copper-D-penicillamine-chloride cluster in aqueous solution and its relevance to the treatment of Wilson's disease.

Complex formation between D-penicillamine (Pen) and copper(II) ions has been studied under simulated physiological conditions in both the presence and absence of the blood plasma constituents albumin, alanine, histidine, and zinc(II). Chromatographic and uv/vis and electron spin resonance (esr) spectroscopic methods were used. The major species formed, at neutral pH and 0.15 mol dm-3 NaCl, is the violet species which is shown to have the same stoichiometry as the recently reported solid-state complex, i.e., [Cu8I Cu6II (Pen)12 Cl] 5-. The rate of formation of this species (MVC) is shown to be dependent on the Cu concentration, Cu:Pen ratio, relative Cl- ion concentration, pH, and temperature. Formation is inhibited by the presence of O2 and biological chelates. At the concentration levels found in blood plasma it is unlikely that the MVC ion has any significance in the therapeutic action of penicillamine in the treatment of Wilson's disease. Reexamination of the aqueous Cu-albumin-pen system reinforces earlier findings that pen is unable to mobilize Cu that is bound to albumin. Significant binding of pen to the protein is observed is not related to any protein-bound copper ions. Evidence that ternary complexes of the type amino acid-Cu-Pen can form in blood plasma is presented. These are unlikely, however, to be physiologically significant and the copper depletion induced by Pen in Wilson's disease cases must be elsewhere than in the blood plasma compartment.

Synthesis of the native copper(II)-transport site of human serum albumin and its copper(II)-binding properties.

A derivative of the native-sequence tripeptide of the specific Cu(II)-transport site of human serum albumin, L-aspartyl-L-alanyl-L-histidine N-methylamide, was synthesized, and its binding to Cu(II) was examined to determine the influence of the side-chain groups on the Cu(II) binding. The equilibria involved in the Cu(II)-L-aspartyl-L-alanyl-L-histidine N-methylamide system were investigated by analytical potentiometry. Three complex species were found in the pH range 4-10. The same species were identified in both the visible and circular-dichroism spectra. The main species present in the physiological pH range is shown to have the same ligands around the square-planar Cu(II) ion as those reported for albumin and tripeptides diglycyl-L-histidine and its N-methylamide derivative. The results obtained from competition experiments showed that this tripeptide has a higher affinity towards Cu(II) than has albumin itself. The overall findings are compared with those from albumin. At neutral pH the side chains do not play any important role in the Cu(II) binding, but at low pH the beta-carboxyl group of the N-terminal aspartic residue becomes important. A possible competition site on albumin for Cu(II) at low pH is discussed.