Authors: Achyuthan, Komandoor E.; McClain, Jaime L.; Raj, Dominic

Source: Combinatorial Chemistry & High Throughput Screening, Volume 12, Number 7, August 2009 , pp. 678-689(12)

Publisher: Bentham Science Publishers

Abstract:

There is considerable interest in the oxidative fate of phenols such as p-cresol as environmental pollutants and uremic toxins. We supply a menu of spectroscopic options for the high throughput screening of laccase oxidation of pcresol through multiple modes of detection. Laccase activity was monitored kinetically at pH 4.5 by absorption changes at 250 nm, 274 nm or 297 nm, and in endpoint mode by the bathochromic shift in absorption to 326 nm in 50 mM NaOH. Laccase oxidation of p-cresol was also detected by product fluorescence at 425 nm after excitation at 262 nm or 322 nm in 50 mM NaOH. We optimized the kinetic parameters for p-cresol oxidation (pH optimum 4.5-5.1; 37oC; Km = 2.2 mM) resulting in laccase limits of detection and quantitation of 25 pg/μL and 75 pg/μL, respectively (∼360 pM; 25 ppb). The sensitivity for p-cresol was similar to previously reported values. The small (∼20%) decrease in signal strength after six cycles of excitation over a 3 h period was attributed to photobleaching or photodegradation of the emitter and not due to fluorescence decay (photoinstability). Halide inhibition was characteristic of laccases (IC50 = 25 mM NaCl). A unique advantage of our assay is that laccase catalysis could be interrogated using multi-mode absorption or fluorescence under acidic or basic conditions, in real time or endpoint modes. Orthogonal interrogation facilitates ratiometric analysis enabling high specificity while minimizing interferences during compound library screening. The phenolic alcohol pcresol may be a model for monolignol oxidation. Our studies might find applications in biofuels, to triage dialysis patients, or for the environmental bioremediation of phenols.

Keywords: Laccase; p-cresol; spectroscopy; absorption; fluorescence; high throughput screening

Document Type: Research article

DOI: http://dx.doi.org/10.2174/138620709788923773

Affiliations: 1: Sandia National Laboratories,Biosensors & Nanomaterials Dept., P.O. Box 5800, MS 1425, Albuquerque,NM 87185-1425, USA.

Publication date: 2009-08-01

More about this publication?

• Combinatorial Chemistry & High Throughput Screening publishes full length original research articles and reviews describing various topics in combinatorial chemistry (e.g. small molecules, peptide, nucleic acid or phage display libraries) and/or high throughput screening (e.g. developmental, practical or theoretical). Ancillary subjects of key importance, such as robotics and informatics, will also be covered by the journal. In these respective subject areas, Combinatorial Chemistry & High Throughput Screening is intended to function as the most comprehensive and up-to-date medium available. The journal should be of value to individuals engaged in the process of drug discoveryand development, in the settings of industry, academia or government.

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