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Anal Biochem. 2010 Apr 15;399(2):268-75. doi: 10.1016/j.ab.2009.12.018. Epub 2009 Dec 14.

Use of thermal melt curves to assess the quality of enzyme preparations.

Author information

  • 1Department of Medicine, University of Washington, Seattle, WA 98195, USA. crowther@uw.edu

Abstract

This study sought to determine whether the quality of enzyme preparations can be determined from their melting curves, which may easily be obtained using a fluorescent probe and a standard reverse transcription-polymerase chain reaction (RT-PCR) machine. Thermal melt data on 31 recombinant enzymes from Plasmodium parasites were acquired by incrementally heating them to 90 degrees C and measuring unfolding with a fluorescent dye. Activity assays specific to each enzyme were also performed. Four of the enzymes were denatured to varying degrees with heat and sodium dodecyl sulfate (SDS) prior to the thermal melt and activity assays. In general, melting curve quality was correlated with enzyme activity; enzymes with high-quality curves were found almost uniformly to be active, whereas those with lower quality curves were more varied in their catalytic performance. Inspection of melting curves of bovine xanthine oxidase and Entamoeba histolytica cysteine protease 1 allowed active stocks to be distinguished from inactive stocks, implying that a relationship between melting curve quality and activity persists over a wide range of experimental conditions and species. Our data suggest that melting curves can help to distinguish properly folded proteins from denatured ones and, therefore, may be useful in selecting stocks for further study and in optimizing purification procedures for specific proteins.

Copyright 2009 Elsevier Inc. All rights reserved.

PMID:
20018159
[PubMed - indexed for MEDLINE]
PMCID:
PMC3270315
Free PMC Article

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