We have been studying on a bioluminescence system of an oceanic luminous squid. Symplectoteuthis oualaniensis L. In 1981, Tsuji and Leisman reported that a homogenate of the luminous organ of this squid gave light in the presence of monocations such as Na^+, K^+ and molecular oxygen at pH 7.8. In 1993, we succeeded in the extraction of a photoprotein. named symplectin, responsible for the bioluminescence in a 0.6M KCl solution from the same squid S. oualaniensis collected in Okinawa, Japan. We reported that this photoprotein includes dehydrocoelenterazine(2) as the chromophore which covalently binds with symplectin through a thioether linkage. The major evidence for this chromophore is that mixing of dehydrocoelenterazine(2) into apo-symplectin significantly enhanced the luminescence intensity. The reddish solution of 2 mixed to apo-symplectin solution instantaneously changed into yellowish color. Recently this phenomenon was successfully mimicked between tri-peptide GSH(glutathione) in place of symplectin and a 100%-^<13>C-labeled methoxy-dehydrocoelenterazine(17). The complex structure (18, 19) were determined by NMR (C-H COSY, HMBC and HOHAHA) and Q-TOF-MS/MS measurement. We found that fluorinated dehydrocoelenterazine(21) gave the most stable chromophore complex(22) with cysteine of symplectin analog; GSH(20). The photoprotein (symplectin) can only become soluble in high salt buffer containing KCl higher than 0.6M. The homogenate of the photogenic organ containing symplectin was first washed with 0.4M KCl solution (pH 6.0), and then extracted with 0.6M KCl buffer to give a crude symplectin solution. Addition of aliquot of the photoprotein solution into pH 8.0 buffer gives light, so it is possible to check the luminescence activity of symplectin. A selective hydrolysis occured with trypsin to convert the original 60kDa (symplectin) into 2 products, namely 40kDa and 15kDa. Both 60kDa and 40kDa proteins showed luminescence activity. The 15kDa was not luminescent, but it possessed the same N-terminal amino acid sequence as the 60kDa symplectin. This means the active center of symplectin exists in the 40kDa site. We have attempted to obtain the cDNA encoding the squid 60kDa symplectin by the cloning procedure based on the polymerase chain reaction (PCR). Recently, we have succeeded in cloning the cDNA and obtaining the amino acid sequence of the 60kDa symplectin. At the same time, some part of the sequence of symplectin has been deduced by means of enzymatic hydrolysis followed by nano-LC-Q-TOF-MS/MS analysis. The complete amino acid sequence of symplectin was obtained by using MS/MS analysis and cDNA. Symplectin has 11 cysteine residues in the total sequence and 8 cysteine residues in the active 40kDa site. One of the cysteine in the 40kDa site must be the active center of bioluminescence activity. The detection of the active site of the symplectin and the investigate molecular mechanisms on the interaction of the chromophore with the apo-symplectin are now in progress in our laboratory.
