정제된 꽃게(Portunus trituberculatus) 헤모시아닌에 CNBr을 처리한 후 전기영동을 한 결과 분자량이 각각 31,000, 19,000, 13,000, 6,000 및 4,000인 다섯 개의 peptide로 절단되었다. 이들을 전기영동도에 따라서 peptide C1부터 C5로 각각 명명하였다. CNBr로 처리된 헤모시아닌을 Sephadex G-50에서 5% propionic acid로 용출할 경우 3개의 분획으로 분리되었고 이들 각 분획에 대한 전기영동 결과 첫번째 분획에는 peptide C1, C2, C3가 응집된 상태로 용출되었고, 두번째 분획에서는 peptide C4와 소량의 C2, C3가, 세번째 분획에서는 peptide C5가 각각 존재함을 확인하였다. Peptide C5는 $Cu^{++}$가 결합된채 분리되었다. Sephadex G-50 에서 용출된 각 분획을 DEAE-cellulose column chromatography를 이용하여 peptide C4와 peptide C5를 순수분리하였다. 순수분리된 peptide C4와 peptide C5 및 꽃게 헤모시아닌에 대한 아미노산 조성을 본 결과 꽃게 헤모시아닌은 Aspartic acid와 Glutamic acid 함량이 높은 것으로 밝혀졌다. Peptide C4의 경우는 몇 개 아미노산을 제외하고는 꽃게 헤모시아닌과 큰 차이가 없었으나 $Cu^{++}$를 함유하고 있는 peptide C5는 Lysine 함량이 특히 높은 강한 염기성으로 나타냈다.
Purified hemocyanin of the crab, Portunus Trituberculatus was treated with CNBr. The SDS electrophoresis of CNBr-treated hemocyanin showed five distinct peptides fragment, referred to as peptide C1, C2, C3, C4, and C5, respectively, according to the mobility in electrophoresis gel. Molecular weights of each peptide estimated by SDS polyacrylamide gel electrophoresis were 31,000, 19,000, 13,000, 6,000, and 4,000, respectively. In order to separate peptides, CNBr-treated hemocyanin was eluted with 5% propionic acid on Sephadex G-50 column chromatography, in which three fractions were obtained. Fraction I contained peptide C1, peptide C2, peptide C3. Peptide C4 was eluted in fraction II with some impurity of peptide C2 and C3, and peptide C5 in fraction III, respectively. Peptide C5 was the smallest fragment and showed the copper-binding property. The amino acid compositions of the whole hemocyanin and CNBr fragments (peptide C4 and peptide C5) have been analyzed. The amino acid composition of whole hemocyanin showed that P. trituberculatus hemocyanin was typically acidic protein. While the amino acid composition of peptide C4 was similar to that of whole hemocyanin, peptide C5, containing copper, showed high content of lysine residue.
