Exposure to high levels of arsenic can cause a wide range of health effects, including cancers of the bladder, lung, skin, and kidney. However, the mechanism(s) of action underlying these deleterious effects of arsenic remains unclear. Arsenic binding to cellular proteins is a possible mechanism of toxicity, and identifying such binding is analytically challenging because of the large concentration range and variety of proteins. We describe here an affinity selection technique, coupled with mass spectrometry, to select and identify specific arsenic-binding proteins from a large pool of cellular proteins. Controlled experiments using proteins either containing free cysteine(s) or having cysteine blocked showed that the arsenic affinity column specifically captured the proteins containing free cysteine(s) available to bind to arsenic. The technique was able to capture and identify trace amounts of bovine biliverdin reductase B present as a minor impurity in the commercial preparation of carbonic anhydrase II, demonstrating the ability to identify arsenic-binding proteins in the presence of a large excess of non-specific proteins. Application of the technique to the analysis of subcellular fractions of A549 human lung carcinoma cells identified 50 proteins in the nuclear fraction, and 24 proteins in the membrane/organelle fraction that could bind to arsenic, adding to the current list of only a few known arsenic-binding proteins.

Fourier transform infrared (FT-IR) spectra have been measured for defatted bovine serum albumin (BSA) in D(2)O with a concentration of 2.0 wt % over a temperature range of 26-90 degrees C and the corresponding difference spectra have been calculated by subtracting the contribution of D(2)O at the same temperature. Evolving factor analysis (EFA) by selecting two factors and three factors has been employed to analyze the temperature-dependent difference IR spectra in the 1700-1600 cm(-1) spectral region of the defatted BSA in D(2)O solution. Three-factor EFA has been employed to determine the distinction of the three protein species involved in the process of temperature elevation: native, transitional, and denatured protein. The temperature profiles obtained from three-factor EFA indicate that heat-induced conformational change in the secondary structures of defatted BSA in D(2)O undergoes two two-state transitions, a drastic transition and a slight transition, which occur in the temperature ranges of 68-82 degrees C and 56-76 degrees C, respectively.

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