JBC, Vol. 254, Issue 3, 769-780, Feb, 1979
Ca2+-induced bioluminescence in Renilla reniformis. Purification and characterization of a calcium-triggered luciferin-binding protein
H. Charbonneau and M. J. Cormier
A Ca2+-triggered luciferin-binding protein (BP-LH2) from the bioluminescent
marine coelenterate, Renilla reniformis, has been purified by conventional
methods. One kilogram of processed animals yields approximately 2.7 mg of
pure protein with an overall yield of 55%. Physicochemical studies show
that BP-LH2 is a globular protein containing one single polypeptide chain
with one disulfide bond. Ultracentrifugation studies, amino acid analysis,
and sodium dodecyl sulfate-gel electrophoresis show that BP-LH2 has an
average molecular weight of 18,500. BP-LH2 has a Stokes radius of 23 A, a
sedimentation coefficient, S020,w, of 2.3 S, and an isoelectric point of
4.3. The acidic nature of the protein was confirmed by amino acid analysis,
which showed that 27% of the residues are acidic. The protein contains no
carbohydrate, phosphate, or tryptophan. There is one noncovalently bound
molecule of coelenterate type luciferin resulting in distinct protein
spectral properties with absorption maxima at 276 nm (epsilon 0.1% 276 =
1.31) and 446 nm (episoln 0.1% 446 = 0.47) and a fluorescence emission at
520 nm (uncorrected). In the presence of Ca2+, BP-LH2 will react with
Renilla luciferase to give the characteristic in vitro blue
bioluminescence. Ca2+ binding produces a distinct change in the spectral
properties of BP-LH2 including a 4-fold enhancement of tyrosine
fluorescence at 332 nm and a 5-fold fluorescence enhancement at 520 nm. In
addition, the visible absorption maximum shifts from 446 nm to 420 nm. The
fluorescence enhancement at 320 nm occurs over the range from 1 to 10
micrometer Ca2+. BP-LH2 has two Ca2+-binding sites with an estimated Kd of
0.02 micrometer, in 10 muM Tris at pH 7.2. BP-LH2 was compared to several
well studied Ca2+-binding proteins and was found to possess similar
Ca2+-binding and physicochemical properties. This study clearly
demonstrates that BP-LH2 is capable of triggering a bioluminescent flash in
response to an intracellular Ca2+ transient.
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