25 trademark column chromatography 100 u00d7 10 u03c6 mm to obtain the objective apoaequorin the apoaequorin was produced using a plasmid which can effect apoaequorin expression in escherichia coli preparation of apoaequorin is disclosed in japanese patent application nos sho 60 280259 1985 sho 61 249098 1986 sho 61 245108 1986 sho 61 245109 1986 sho 62 126373 1987 sho 62 126374 1987 and biochemistry 1986 25 8425 8429 inoue et al or apoaequorin is obtained by further purifying the above apoaequorin according to anion exchange chromatography deae cellulofine trademark deae sephacel trademark etc to a solution 2 ml of apoaequorin 100 u03bcg is added 2 mercaptoethanol 100 u03bcl capable of effecting scission of the disulfide bond s s bond of protein followed by allowing the mixture to stand at 4 u00b0 c for one hour or longer to prepare a reduction type apoaequorin the reduction type apoaequorin 2 u03bcl prepared in the above item 2 is mixed with a substrate coelenterazine 2 u03bcl 100 u03bcg ml followed by setting the mixture in a reaction cuvette of a lumiphotometer td4000 type trademark of an instrument made by laboscience company tokyo pouring 30 mm cacl 2 solution 100 u03bcl therein or pouring 30 mm cacl 2 solution 100 u03bcl in the reduction type apoaequorin 2 u03bcl mixing these pouring a substrate coelenterazine 2 u03bcl in the mixture and recording a continuously emitted light in.