Vanadyl (IV) and vanadate (V) binding to selected endogenous phosphate, carboxyl, and amino ligands; calculations of cellular vanadium species distribution

Abstract Vanadium enters cells as vanadate (V) where it is reduced to vanadyl (IV), VO 2+ . Vanadate species at plasma pH, H 2 VO 4 − , and HVO 4 2− are referred to as VO 3 − . To gain an insight into the subcellular vanadium distribution we measured the binding of VO 3 − and VO 2+ to extra- and intracellular ligands, and calculated free and bound fractions of these ions for expected in vivo conditions. The association constants ( K ) were determined by the pH shift caused by an addition of VOSO 4 or NaVO 3 to individual ligand solutions at 20 °C and a pH equal to the p K of the reactive groups. The p k 's for binding of VO 2+ were ATP, 5.9; ADP, 5.5; AMP, 5.1; P i 4.3; creatine phosphate (CP), 3.6; glutamic acid, 3.4; aspartic acid, 3.1; human serum albumin, 3.1; glutathione, 2.7; ascorbic acid, 3.3; citric acid, 4.0. The p k of VO 3 − and human serum albumin was 3.3 and of that VO 3 − and glutathione was 4.2. VO 3 − did not bind to ATP, even via Mg 2+ or Ca 2+ bridges. We calculated that in cells ~1% of total VO 2+ is unbound, which is 10 −10 –10 −9 m since published values for total vanadium (mainly VO 2+ ) concentrations in tissues are on the order of 10 −8 –10 −7 m . Free VO 2+ may be even less because of binding to additional ligands not considered and due to spontaneous hydrolysis to VOOH + and VO(OH) 2 2+ at intracellular pH. The binding of VO 2+ to each ligand was corrected for presence of multiple ligands and competition by H + , K + , and Mg 2+ . In cells with no CP, up to 70% of VO 2+ is bound to phosphates and up to 29% to proteins; in cells with 30 m m CP (as in muscle), ~95% is bound to phosphates (CP binds up to 61% of total VO 2+ ) and ~4% to proteins; in cells with 2 m m ascorbic acid (as in brain), the vitamin binds ~3% of total VO 2+ . These binding values apply for the total VO 2+ concentration range of 10 −8 –10 −5 m . The intracellular binding and a reducing environment protect the freshly reduced VO 2+ from oxidation to VO 3 − that would otherwise occur at neutral pH. This strong affinity of VO 2+ primarily for phosphates also explains the mechanism for the intracellular accumulation of vanadium which is a factor in previously observed transport of VO 3 − into cells. Given 90% protein binding, free plasma VO 3 − would be ~10 −9 m , since total vanadium (mainly VO 3 − ) concentration in human plasma is ~10 −8 m . Passive passage against the electrochemical gradient, calculated by the Nernst equation, would result in 3 × 10 −11 m free VO 3 − inside the cell, a concentration ~100-fold lower than the published dissociation constant of VO 3 − and the high affinity site of the (Na + + K + )-ATPase molecule. Thus, given cytoplasmic site of action of vanadium and absence of an active transport, normal intracellular concentrations of free VO 3 − and VO 2+ appear to be too low for the regulation of the (Na + + K + )-pump. ATP deficiency causes redistribution of VO 2+ to other ligands but insufficient free VO 2+ is liberated to affect known susceptible enzymes. On the other hand, overdosing with VO 3 − is known to result in reduced (Na + + K + )-ATPase activity and renal damage.