Conformational Heterogeneity Of Homologous Azurins And Their Metallo-Derivatives: A Time-Resolved Fluorescence Study

Cindy M.L Hutnik; Arthur G Szabo

doi:10.1117/12.945405

24 June 1988 Conformational Heterogeneity Of Homologous Azurins And Their Metallo-Derivatives: A Time-Resolved Fluorescence Study

Cindy M.L Hutnik, Arthur G Szabo

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Proceedings Volume 0909, Time-Resolved Laser Spectroscopy in Biochemistry; (1988) https://doi.org/10.1117/12.945405
Event: 1988 Los Angeles Symposium: O-E/LASE '88, 1988, Los Angeles, CA, United States

Abstract

The azurins are a family of homologous blue copper proteins which function as bacterial electron transferases (1). These proteins have been actively studied owing to several of their unusual and remarkable spectroscopic and chemical properties (2). In this study, homologous azurins from Pseudomonas fluorescens (ATCC 13525) and Pseudomonas aeruginosa (ATCC 10145) were purified and examined by a number of electrophoretic techniques and their copper:protein stoichiometry determined by atomic absorption and amino acid analysis. Provided that the spectral ratios (A620/A280) were above 0.50 and no evidence of a Soret band in the absorption spectrum existed, results showed that there was no contamination of these blue-copper proteins with either cytochrome or a "copper-less" apoazurin. Upon isoelectric focusing for example, apoazurin clearly migrated to a more acidic position relative to the holoazurin and only when the spectral ratio was below 0.50 could an extra band, co-migrating with apoazurin, be detected in holoazurin samples. Collectively the above results not only provided convincing evidence of protein homogeneity but also established, for the first time, definite criteria by which to judge azurin homogeneity.

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Cindy M.L Hutnik and Arthur G Szabo "Conformational Heterogeneity Of Homologous Azurins And Their Metallo-Derivatives: A Time-Resolved Fluorescence Study", Proc. SPIE 0909, Time-Resolved Laser Spectroscopy in Biochemistry, (24 June 1988); https://doi.org/10.1117/12.945405

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KEYWORDS

Proteins

Luminescence

Copper

Absorption

Time resolved spectroscopy

Biochemistry

Metals

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