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HPLC를 이용한 신기능성 고분자, 폴리감마 글루탐산의 입체구조 조성비의 정량
유동찬,지광환 금오공과대학교 2005 論文集 Vol.26 No.-
Poly-v-glutamic acid (γ-PGA) is an unusual anionic polypeptide in which D-and/or L-glutamate is polymerized via γ-amide linkages. Reversed-phase high-performance liquid chromatography and pre-column derivation with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marley's reagent) were used for assaying the stereotype composition of L and D-glutamic acid followed by the total hydrolysis of γ-PGA The determination of the stereotype ratio for the γ-PGA is important for the application for its industrial use. Our rrethod is more convenient and reproducible compared with the previous method that is using chiral colurrm for the separation of the racemates of amino acids. Our data obtained with HPLC method exhibited good quantitative correlation with that from enzymatic analysis of γ-PGA Our data would be a useful and convenient method for quality control for the mass production of γ-PGA.
γ-PGA에 포함된 D-glutamate의 효소화학적 정량분석
조현정,지광환 금오공과대학교 2005 論文集 Vol.26 No.-
Bacillus subtilis chungkookjang produces a new biofunctional polymer, poly y-glutamic acid (γ-PGA). γ-PGA consists of D and/or L-glutamate and its ratio is different depending microorganisms and culturing environrrents, Here we are reporting the new enzymatic method to determine the stereochemistry of D-glutamic acid composition ratio in γ-PGA D-amino acid aminotransferase (D-AAT) can catalyzes transamination between D-amino acids and a-keto acids. Thus the amount of D-glutamate in γ-PGA can be determined by the assaying the amount of produced D-alanine followed by the reaction of D-AAT in the presence of pyruvic acid. D-glutamate and D-alanine can be separated by thin layer chromatography (TLC) in NH₄0H : ethanol (2:8, v/v) solvent system D-alanine can be visualized by ninhydrin (0.5% in 75% ethanol) spraying and quantitated by the measurement the absorbance of TLC spot followed by the elution with 0.005% copper sulfate in 75% ethanol at EOOnm. Partially purified D-AAT is good enough to carry out this new and very convenient method to determine the amount of D-glutamic acid in γ-PGA.
Homocysteine-thiolactone 이 유발시키는 소 혈관내피세포의 손상효과
유동찬,김보은,전가영,권오형,지광환 금오공과대학교 2007 論文集 Vol.28 No.-
The homocysteine-thiolactone, a reactive metabolic compound of homocysteine has been implicated in human cardiovascular diseases. The increasing of total plasma homocysteine concentration lead to homocystinuria, which may cause to elevate the concentration of homocysteine-thiolactone in the blood serum. It might be one of the risk factor which is responsible for the development of several vascular diseases. The present study is to demonstrate the harmful effect of homocysteine-thiolactone induced on calf pulmonary artery endothelial (CPAE) cells. CPAE were cultured in RPMI1640 medium supplemented with 10% FBS. CPAE were grown in humidified atmosphere of 5% C₂ in air at 3TC. Cells cultured with homocysteine-thiolactone at concentrations ranging from 0.1mM, lmM, 5mM and l0mM (dissolved in dH₂O). After incubation, cells were stained with MTT. Cell viability was evaluated under the UV-visible absorbance at 540nm. Cells viability was significantly decreased by increasing the concentration of homocysteine-thiolactone, Our data provide the evidence to understand the effect of homocysteine-thiolactone on the cardiovascular system.