using MS/MS strategies. As it was established that Cysteine residues have a high affinity for trivalent Arsenic, ESI-MS revealed that Arsenite was unable to bind to haemoglobin and thioredoxin after cysteine residues were blocked with N-ethylmaleimide (NEM)
In rat hemoglobin, trivalent Arsenic was found to bind to a preferentially specific cysteine (Cys13) employing ESI-MS. This cysteine13 was found to be present in a hydrophobic pocket providing a microenvironment for DMAIII.
5.2. Binding Stoichiometry
A well defined structure of metal-protein interactions are needed for X-ray crystallography technique. Further a sufficient amount of ligand needs to be available in order to form a completely occupied stoichiometric structure. This complex can be obtained by drenching the ligand into the crytal or by cocrystallization. The binding metal ions are yielded in integers during mass spectrometry analysis though all the proteins may not be loaded with metal ions while as isothermal calaorimetry and titration based methods give noninteger numbers of metal ions binding to proteins. The Arsenite binding stoichmetries were found to be consistant in proteins like hemoglobin and metallothionein while employing techniques like MALDI-MS or ESI-MS.
5.3. Binding Affinity
The changes in the protein/peptide properties can be used to find the binding affinity dissociation constant (Kd) and association constant (Ka). The procedures employed to find the binding affinities include intrinsic tryptophan fluorescence quenching, absorbance shift of arsenical or protein/peptide, capillary electrophoresis, measuring the reduction of thiol content after etc. In an alternate method Arsenic can be partitioned into protein bound form and free form at the binding equilibrium by separating the two forms using molecular sieve chromatography, filiteration or dialysis. Fluoresence labelled Arsenic and radioactivity has also been used to find binding affinities of Arsenic-protein binding. Isothermal titration calorimetry is an efficient technique to measure multiple parameters like binding enthalpy and entropy, binding stoichiometry and binding constant.
The problem with measuring the unbound and bound species with ESI-MS is the different ESI-MS responses of these species and the intensities may not be caliberated against a single species. Therefore it is important to measure relative rather than absolute values using ESI-MS.
Inhibition study is also an important tool to find the effect of arsenic on enzyme activity. When inhibition studies are employed to measure dissociation constant of the inhibitor it is known as inhibition constant Ki. It was further found that inhibition of enzymes by Arsenic can be either competitive or uncompetitive.
The Arsenic toxicity being a global phenomenon is major issue in public health and therefore an intense research is warranted. Many tpes of cancers like lungs, bladder, skin etc have been confirmed to be a consequence of chronic Arsenic exposure while for other cancers like kidney cancer, prostrate cancer and leukemia, the data is limited. The susceptibility of humans to diabetes on Arsenic exposure is not well understood. .
The biological affects of Arsenic are so diverse that multiple mechanisms have been proposed for the toxicity of the Arsenic. The mechanisms involved in the Arsenic induced carcinogenesis are also diverse and complicated.. The interaction of Arsenic with sulfhydryl groups in proteins is considered one of the principle mechanisms which triggers the cellular