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An electrospray ionization mass spectrometry investigation of 1-anilino-8-naphthalene-sulfonate (ANS) binding to proteins

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Journal of the American Society for Mass Spectrometry

Abstract

The binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to various globular proteins at acidic pH has been investigated by electrospray ionization mass spectrometry (ESI-MS). Maximal ANS binding is observed in the pH range 3–5. As many as seven species of dye-bound complexes are detected for myoglobin. Similar studies were carried out with cytochrome c, carbonic anhydrase, triosephosphate isomerase, lysozyme, α-lactalbumin, and bovine pancreatic trypsin inhibitor (BPTI). Strong ANS binding was observed wherever molten globule states were postulated in solution. ANS binding is not observed for lysozyme and BPTI, which have tightly folded structures in the native form. α-Lactalbumin, which is structurally related to lysozyme but forms a molten globule at acidic pH, exhibited ANS binding. Reduction of disulfide bonds in these proteins leads to the detection of ANS binding even at neutral pH. Binding was suppressed at very low pH (<2.5), presumably due to neutralization of the charge on the sulfonate moiety. The distribution of the relative intensities of the protein bound ANS species varies with the charge state, suggesting heterogeneity of gas phase conformations. The binding strength of these complexes was qualitatively estimated by dissociating them using enhanced nozzle skimmer potentials. The skimmer voltages also affected the lower and higher charge states of these complexes in a different manner.

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Authors and Affiliations

  1. Molecular Biophysics Unit, Indian Institute of Science, 560012, Bangalore, India

    Soumya S. Ray, S. Kumar Singh & P. Balaram

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  1. Soumya S. Ray
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  2. S. Kumar Singh
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  3. P. Balaram
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Correspondence to P. Balaram.

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Ray, S.S., Singh, S.K. & Balaram, P. An electrospray ionization mass spectrometry investigation of 1-anilino-8-naphthalene-sulfonate (ANS) binding to proteins. J Am Soc Mass Spectrom 12, 428–438 (2001). https://doi.org/10.1016/S1044-0305(01)00206-9

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  • DOI: https://doi.org/10.1016/S1044-0305(01)00206-9

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