Semi-synthetic aequorin. An improved tool for the measurement of calcium ion concentration.
The photoprotein aequorin isolated from the jellyfish Aequorea emits blue light in the presence of Ca2+ by an intramolecular process that involves chemical transformation of the coelenterazine moiety into coelenteramide and CO2. Because of its high sensitivity to Ca2+, aequorin has widely been used as a Ca2+ indicator in various biological systems. We have replaced the coelenterazine moiety in the protein with several synthetic coelenterazine analogues, providing semi-synthetic Ca2+-sensitive photoproteins. One of the semi-synthetic photoproteins, derived from coelenterazine analogue (II) (with an extra ethano group), showed highly promising properties for the measurement of Ca2+, namely (1) the rise time of luminescence in response to Ca2+ was shortened by approx. 4-fold compared with native aequorin and (2) the luminescence spectrum showed two peaks at 405 nm and 465 nm and the ratio of their peak heights was dependent on Ca2+ concentration in the range of pCa 5-7, thus allowing the determination of [Ca2+] directly from the ratio of two peak intensities. Coelenterazine analogue (I) (with a hydroxy group replaced by an amino group) was also incorporated into apo-aequorin, yielding a Ca2+-sensitive photoprotein, which indicates that an electrostatic interaction between the phenolate group in the coelenterazine moiety and some cationic centre in apo-aequorin is not important in native aequorin, contrary to a previous suggestion.
Shimomura
O
O
Marine Biological Laboratory, Woods Hole, MA 02543.
Musicki
B
B
Kishi
Y
Y
D000331:Aequorin / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D000818:Animals; D002118:Calcium / Q000032:analysis*; D007093:Imidazoles; D007700:Kinetics; D008163:Luminescent Measurements; D008164:Luminescent Proteins; D011719:Pyrazines; D037401:Scyphozoa; D013053:Spectrophotometry
D007093:Imidazoles; D008164:Luminescent Proteins; D011719:Pyrazines; C017144:coelenterazine; D000331:Aequorin; D002118:Calcium