Inactivation of firefly luciferase with N-(iodoacetyl)-N'- (5-sulfo-1-naphthyl)ethylenediamine (I-AEDANS).
N-Iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (I-AEDANS), a fluorescent reagent that selectively modifies cysteine residues, was demonstrated to irreversibly inhibit native Photinus pyralis luciferase purified from firefly lanterns. Complete inactivation of luciferase activity was accompanied by the blockage of all four cysteine thiols and the concomitant incorporation of 4 mol of N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) per mole of enzyme. Employing proteolytic digestions of AEDANS-labeled luciferase and reverse-phase-high-performance liquid chromatography (RP-HPLC), seven tagged peptides were isolated. The AEDANS label provided a convenient spectroscopic marker for the identification of the modified peptides. The sequences of the labeled peptides were deduced from electrospray ionization mass spectrometry (ESMS) and N-terminal sequencing. The fluorescent peptides included cysteine residues and spanned sequences composed of amino acids Leu78-Lys85, Thr214-Arg218, Asp224-Arg275, and Gly388-Met396. The luciferin substrate provided substantial protection against luciferase inactivation resulting in a 60-67% decrease in the labeling of all four cysteine thiols. Thus, it does not appear that a specific cysteine mediates the loss of luciferase activity. Additional LC/ESMS studies permitted the identification of 78% of the native luciferase molecule, which, unlike the recombinant protein, was found to contain an acetylated N-terminus. The AEDANS labeling results and the identification of well-defined proteolytic fragments should facilitate future structure-function investigations of the firefly luciferases.
Branchini
B R
BR
Department of Chemistry, Connecticut College, New London 06320, USA. brbra@conncoll.edu
Magyar
R A
RA
Murtiashaw
M H
MH
Magnasco
N
N
Hinz
L K
LK
Stroh
J G
JG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001517:Coleoptera / Q000201:enzymology; D003545:Cysteine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D049454:Firefly Luciferin / Q000378:metabolism; D008156:Luciferases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000494:pharmacology*; D010449:Peptide Mapping; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014357:Trypsin
D004791:Enzyme Inhibitors; D009282:Naphthalenesulfonates; D013439:Sulfhydryl Reagents; D049454:Firefly Luciferin; C006421:1,5-I-AEDANS; D000255:Adenosine Triphosphate; D008156:Luciferases; D014357:Trypsin; D003545:Cysteine