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강옥주,서명자,이안종,김세권,Kang, Ok-Ju,Suh, Myung-Ja,Lee, An-Jong,Kim, Se-Kwon Korean Society of Life Science 1995 생명과학회지 Vol.5 No.4
Carotenoids liberated from the red carotenoprotein from the skin of ascidian(Halocynthia roretzi)were developed on TLC, and two bands were obtained with different Rfvalues. Their chemical reactivities and spectroscopic properties were studied and elucidated as astaxanthin diester and astaxanthin monoester. After saponification of carotenoid ester, GC analysis was performed. The carotenoid ester contained oleic acid, palmitic acid and palmitoleic acids as 50% of total amount, and had higher content unsaturated fatty acid including eicosapentanoic acid than saturated fatty acid.
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강옥주,서명자,이안종,김세권,Kang, Ok-Ju,Suh, Myung-Ja,Lee, An-Jong,Kim, Se-Kwon Korean Society of Life Science 1995 생명과학회지 Vol.5 No.4
A carotennnoprotein from the skin of Ascidian(Halocynthia roretzi) was extracted by Triton X-100 and purified by ammonium sulfate fraction, SephadexG-200 charomatography and DEAE-cellulose ion exchange chromatography. The carotenoprotein was redwith broad $\lambda$$_{max}$ between 495, 467 and 318nm. The red carotenoprotein had an approximate molecular weight of 326KDa(gel filtration). SDS-PAGE indicated the presence of two polypeptodes of 84.1KDa and 74.4KDa, with different mobility in polyacrylamide gel electrophoresis. In the presence of denaturing agents such as organic solvent aand extreme pH, the red complex readily disociates to liberate the yellow carotenoid($\lambda$$_{max}$ 452nm) and a colourless apoprotein. The amino acid composition of carotenoprotein were mainly threonine(15.2%), aspartic acid(12.2%), glutamic acid(11.9%) and serine(9.6%), while proline was not found. The carotenoprotein consisted of lipids as structure units. Its major fatty acids composion were C$_{18:1}$, C$_{16:1}$, and C$_{16:0}$. The monounsaturated fatty acids(41.5%) contained abundant content compared to other fatty aacids(polyunsaturated fatty acids 37.4%, saturated fatty acids 20.6%).
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진주담치 근육중의 적색 Carotenoprotein 의 정제 및 특성
김용태,김세권,양현필,이안종 한국수산학회 1994 한국수산과학회지 Vol.27 No.5
우리나라에서 대량 생산되고 있는 진주담치 근육에서 Triton X-100를 사용하여 carotenoprotein을 추출, 정제하여 그 특성을 실험한 결과를 요약하면 다음과 같다. Carotenoprotein(λ_(max)=462㎚)를 20% (w/v) (NH₄)₂SO₄, DEAE-cellulose 이온교환수지 그리고 Sephacryl S-300 겔 크로마토그래피를 통해 정제한 결과, 정제도는 5배 증가하였고 수율은 1.85%였으며 , carotenoprotein의 분자량은 약 372KDa(gel filtration)이었고, SDS-PAG 전기영동으로 단백질의 subunit를 확인한 결과, 60KDa(23.70%), 46.9KDa(9.14%), 26KDa(49.14%) 및 13KDa(18.02%)로 이루어져 있었다. Carotenoprotein의 안정성은 pH 4.0이하의 강산 및 pH 10.0 이상의 강알칼리에서는 단백질이 불안정하였고, SDS 변성제를 0.2%(w/v)되게 첨가하여 녹인 후 가시부 영역(400∼600㎚)에서 최대파장 변화를 보면 462㎚에서 456㎚로 약 6㎚ 단파장 영역으로 이동한 것을 알 수 있었다. Carotenoprotein의 구성아미노산 조성은 threonine이 35.90%로 다른 아미노산에 비해 함량이 높았으며, 필수아미노산과 소수성 아미노산의 함량은 전체아미노산 함량의 각각 62.76%와 25.02%를 차지하였고, methionine, histidine 및 proline은 존재하지 않았다. Carotenoprotein 1g당 총지질의 함량은 256㎎였고, 그 구성지방산의 조성은 polyene산이 62.4%로 가장 많았고 다음으로 포화산(19.58%) 및 monoene산(18%) 순이었다. 특히, 16:4n-3의 조성비가 33.90%로 상당히 높게 나타났고, EPA, DHA 등 ω3 계의 장쇄(長鎖)고도불포화지방산의 조성비가 22.13%로 높게 나타났으며, 양적으로 많은 지방산은 포화산 중에는 Palmitic acid(C_(16:0)), monoene산 중에서는 palmitoleic acid(C_(16:1)), polyene산 중에서는 hexadecatetraenoic acid(C_(16:4)), eicosapentaenoic acid(C_(20:5)), docosahexaenoic acid(C_(22:6)) 등이 함량이 많았다. Carotenoprotein으로부터 분리한 총 caroteoid 함량은 8.80㎎% 였고, 주 carotenoid는 mytiloxanthin(74.79%), 3,4,3′-trihydroxy-7′,8′-didehydro-β-carotene(18.26%)였다. Most of carotenoprotein complexes have been extracted by using buffered solutions. However, in this study carotenoprotein from the muscle of Blue mussel (Mytilus edulis) was extracted by a detergent such as Triton X-100. It was purified and characterized by 20%(w/v)(NH₄)₂SO₄, DEAE-cellulose ion exchange and Sephacryl S-300 gel filtration. The carotenoprotein(λ_(max)=462㎚) had an approximate M. W. of 372KDa(gel filtration). SDS-PAGE analysis of the carotenoprotein indicated the presence of four polypeptides of 60KDa(23.70%), 46.9KDa(9.14%), 26KDa(49.14%) and 13KDa(18.02%). Carotenoprotein denaturated by treatment with SDS to a final concentration of 0.2%(w/v) caused a hypsochromic shift of λ_(max) from 462㎚ to 456㎚. The carotenoprotein contained lipids as structure units. The amino acid composition of the carotenoprotein contained large essential amino acid amounts of 62.8 % , and the content of threonine(35.9 % ) was higher than other amino acids, but histidine, methionine and proline were not present. In the carotenoprotein, the major fatty acids were C(16:4), C_(16:0), C-(20:5) and C_(22:6). The percentages of polyunsaturated fatty acids(62.4%) were higher compared to other fatty acids(saturated fatty acids 19.6%, monounsaturated fatty acids 18.0%). Carotenoid was extracted from the carotenoprotein by acetone and it was separated into five different components by preparative TLC(benzene:petroleum ether:acetone=69:17:14). The major components of carotenoid were mytiloxanthin(74.79%) and 3,4,3′trihydroxy-7′,8′-didehydro-β-carotene(18.26%), and they were at least presented as prosthetic groups of carotenoprotein.
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