Studies of metal binding and enzyme inhibition by salivary histatin 5
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Histatins are small histidine-rich salivary polypaptides, which exhibit antimicrobial activity against Candida ablicans. This anti-microbial activity has been ascribed in part to a high content of basic amino acids. However, unlike most other antimicrobial proteins histatins have a relatively high content of histidines, tyrosines and acidic amino acids, which participate in metal ion coordination. Therefore, the first aim of this investigation was to determine whether histatins could associate with metal ions, which are known to be present in salivary secretions and whole saliva. In a spectrophotometric competition assay using the metalochromic indicator murexide, it was found that histatin 3 and histatin 5 dissociated complexes of murexide with zinc and copper. Complexes with nickel and cobalt were also dissociated, but to a lesser extent. In similar experiments, it was found that histatin 1, histatin 3 and histatin 5 were able to dissociate complexes of methylthymol blue with calcium and magnesium. Physical binding parameters and spectral properties of zinc- and copper-histatin complexes were investigated to obtain direct evidence of these interactions. Absorption spectra of histatin 5 at increasing copper chloride concentrations resulted in higher absorbance in the 230 to 280 m wavelength range and this spectral change was saturated at a peptide:metal molar ratio of approximately 1:1. A corresponding band was observed in the visible range of the spectrum with an absorbance maximum and molar extinction coefficient corresponding to that of a well known copper binding motif (ATCUN: Asp-Ser-His). Quantitative assessment of zinc and copper binding to histatin 5 using isothermal titration calorimetry revealed at least one high affinity site for zinc and copper, with binding constants of 1.2 x 10[superscipt 5] and 2.6 x 1O[superscript 7] M[superscript -1], respectively. These results indicate that histatin 5 exhibits metallopeptide-like properties. The biological significance of this property could be that histatins contribute to salivary metal binding capacity or interfere with biomolecules that require metals as co-factors. [TRUNCATED]
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Thesis (D.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 2000 (Oral Biology).Includes bibliographical references (leaves 176-192).
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