Use of thermal melt curves to assess the quality of enzyme preparations.
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.
Crowther
Gregory J
GJ
Department of Medicine, University of Washington, Seattle, WA 98195, USA. crowther@uw.edu
He
Panqing
P
Rodenbough
Philip P
PP
Thomas
Andrew P
AP
Kovzun
Kuzma V
KV
Leibly
David J
DJ
Bhandari
Janhavi
J
Castaneda
Lisa J
LJ
Hol
Wim G J
WG
Gelb
Michael H
MH
Napuli
Alberto J
AJ
Van Voorhis
Wesley C
WC
D000818:Animals; D002417:Cattle; D057056:Cysteine Proteases / Q000737:chemistry; D004748:Entamoeba histolytica / Q000201:enzymology; D057075:Enzyme Assays; D004798:Enzymes / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006358:Hot Temperature; D044367:Phase Transition; D010961:Plasmodium / Q000201:enzymology; D011489:Protein Denaturation; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D014969:Xanthine Oxidase / Q000737:chemistry
D004798:Enzymes; D005456:Fluorescent Dyes; D012967:Sodium Dodecyl Sulfate; D014969:Xanthine Oxidase; D057056:Cysteine Proteases