A Characterization of Cysteinyl Groups of Rat Liver Metallothionein

권훈정,고은희,신환철,최명언,Kwon, Hoon-Jeong,Koh, Eun-Hie,Shin, Whan-Chul,Choi, Myung-Un 생화학분자생물학회 1984 한국생화학회지 Vol.17 No.3

메탈로사이오닌(Metallothionein, MT)은 다량의 금속과 결합되어 있는 단백질로, 이를 카드뮴을 주사한 쥐의 간으로부터 분리 정제하였다. 정제과정은 열처리, Sephadex G-50 젤 여과법 및 DEAE Sephadex A-25 이온교환 크로마토그래피법을 포함한다. 두개의 isoprotein인 MT-A와 MT-B를 총회 수율 30%로 분리하였다. MT의 금속 결합부위의 화학적 특성을 DTNB (5, 5'-dithiobis(2-nitrobenzoic acid))를 사용하여 검토해 보았다. 이 단백질과 DTNB의 반응은 pH와 이온세기에 민감하게 변화했으며, pH 변화에 따른 pseudo-first order 반응속도 상수는 pH 4 정도에서 최소치를 보였다. 또한 DTNB에 의해 적정되는 MT 내 thiol의 수가 금속의 존재여부나 단백질 구조변화에 따라 상당히 변화함을 알 수 있었으며, 따라서 이 단백질은 적어도 두 가지 종류의 카드뮴-황 결합상태가 존재하리라고 여겨진다. MT (metallothionein), metal-binding protein, was induced with Cd and isolated from rat liver supernatant by heat-treatment, Sephadex G-50 gel filtration, and DEAE Sephadex A-25 ion exchange chromatography. Two isoproteins, MT-A and MT-B, were separated with overall yield of 30%. In order to characterize the chemical properties of metal-binding sites of MT, the cysteinyl groups were examined with DTNB. The reaction of MT-A with DTNB was dependent on the pH and ionic strength. The plot of pseudo-first order rate constant against pH showed a minimum near pH 7.5. It was also found that the DTNB-titrable thiol groups of MT-A were markedly affected by either the presence of metal or by the changes of protein conformation, thus this protein appears to contain at least two different types of cadmium-sulfur bonding.

쥐간 메탈로사이오닌의 시스테인기의 특성연구

권훈정,고은희,신환철,최명원 ( Hoon Jeong Kwon,Eun Hie Koh,Whan Chul Shin,Myung Un Choi ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.3

MT (metallothionein), metal-binding protein, was induced with Cd and isolated from rat liver supernatant by heat-treatment, Sephadex G-50 gel filtration, and DEAE Sephadex A-25 ion exchange chromatography. Two isoproteins, MT-A and MT-B, were separated with overall yield of 30%. In order to characterize the chemical properties of metal-binding sites of MT, the cysteinyl groups were examined with DTNB. The reaction of MT-A with DTNB was dependent on the pH and ionic strength. The plot of pseudofirst order rate constant against pH showed a minimum near pH 7.5. It was also found that the DTNB-titrable thiol groups of MT-A were markedly affected by either the presence of metal or by the changes of protein conformation, thus this protein appears to contain at least two different types of cadmium-sulfur bonding.

Molecular Characteristics of Cysteinyl Groups of Metallothionein by 5,5'-Dithiobis(2-nitrobenzoic acid)

김봉섭,권훈정,최명언,고은희,Kim, Bong-Sub,Kwon, Hoon-Jeong,Choi, Myung-Un,Koh, Eun-Hie 생화학분자생물학회 1987 한국생화학회지 Vol.20 No.3

In the previous study of the reaction of the cysteinyl groups of metallothionein (MT) with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) (Korean Biochem. J. (1984) 17, 288-298), it was found that the DTNB titrable cysteinyl groups were markedly affected either by the presence of metal or by the changes of protein conformation. In order to clarify these phenomena, we prepared the a fragment (4-metal cluster) of MT-I and examined the DTNB reaction. The ratio of 1.7 was obtained as the DTNB titrable cysteinyl groups to the bound metal ions. Thus these data support the notion that only one type of cysteinyl groups may interact with DTNB, mainly simply coordinated cysteinyl groups. Furthermore, we investigated the effects of var ous disulfides on the DTNB reaction rates to explore the microenvironment around the cysteinyl groups. Disulfides tested were cystamine, 2-hydroxyethyl disulfide, dimethyl disulfide, and 3,3'-dithiodipropanoic acid. The DTNB reaction rate of MT-I was enhanced by cystamine and 2-hydroxyethyl disulfide, but that of the a fragment was inhibited by both disulfides. Others had no effects on the DTNB reaction of MT. Therefore, the DTNB titrable cysteinyl groups of MT seem to be located in a hydrophilic and negatively charged microenvironment. 메탈로사이오닌과 5,5'-dithiobis(2-nitrobenzoate) (DTNB)의 반응연구에 의하면 (Korean Biochem. J. (1984) 17, 288-289), DTNB에 의하여 적정되는 cysteinyl기의 수는 금속의 존재나 메탈로사이오닌의 구조에 영향을 받는다. 이것을 더 연구하기 위해 메탈로사이오닌의 $\alpha$ fragment를 분리하여 DTNB와 반응시켰다. DTNB로 적정가능한 cysteinyl기의 수와 금속이온수의 비는 1.7이었다. 이것은 메탈로사이오닌 내의 cysteinyl키 중 simply coordinate된 것만이 DTNB와 반응함을 뒷받침 해준다. 더 나아가 cysteinyl 거의 분자내 환경을 알기 위해 disulfide 의 영향을 살펴보았다. Cystamine, 2,2'-dihydroxyethyl disulfide, 3,3'-dithiodipropanoic acid, dimethyl disulfide 동의 disulfide가 사용 되었다. 메탈로사이오닌-I의 경우에는 cystamine과 2,2'-hydroxyethyl disulfide에 의하여 반응속도가 증가하였으나 $\alpha$ fragment의 경우에는 감소하였다. 다른 두 disulfide는 반응속도에 영향을 마치지 않았다. 따라서 DTNB와 반응하는 메탈로사이오닌의 cysteinyl기는 그 미세환경이 음성전하를 띠고 친수성의 위치에 처해 있다고 추정된다.

5 ' 5 ' - dithiobis ( 2 - nitrobenzoic acid ) 에 의한 메탈로사이오닌 cystein 기의 분자 특징

김봉섭,권훈정,최명언,고은희 ( Bong Sub Kim,Hoon Jeong Kwon,Myung Un Choi,Eun Hie Koh ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.3

In the previous study of the reaction of the cysteinyl groups of metallothionein (MT) with 5,5`-dithiobis(2-nitrobenzoic acid) (DTNB) (Korean Biochem. J. (1984) 17, 288-298), it was found that the DTNB titrable cysteinyl groups were markedly affected either by the presence of metal or by the changes of protein conformation. In order to clarify these phenomena, we prepared the a fragment (4-metal cluster) of MT-I and examined the DTNB reaction. The ratio of 1.7 was obtained as the DTNB titrable cysteinyl groups to the bound metal ions. Thus these data support the notion that only one type of cysteinyl groups may interact with DTNB, mainly simply coordinated cysteinyl groups. Furthermore, we investigated the effects of var ous disulfides on the DTNB reaction rates to explore the micro environment around the cysteinyl groups. Disulfides tested were cystamine, 2-hydroxyethyl disulfide, dimethyl disulfide, and 3,3`-dithiodipropanoic acid. The DTNB reaction rate of MT-I was enhanced by cystamine and 2-hydroxyethyl disulfide, but that of the α fragment was inhibited by both disulfides. Others had no effects on the DTNB reaction of MT. Therefore, the DTNB titrable cysteinyl groups of MT seem to be located in a hydrophilic and negatively charged microenvironment.

다시마와 미역의 섭취가 발암물질에 의한 DNA 손상과 칼슘 및 철 흡수에 미치는 영향

성미경(Sung Mi-Kyung),한경희(Han Kyung-Hee),권훈정(Kwon Hoon-Jeong),박유경(Park You-Gyung),부소영(Bu So-Young) 韓國營養學會 2000 Journal of Nutrition and Health Vol.33 No.7

재래식 간장의 제조조건이 ethyl carbamate 생성에 미치는 영향

권훈정,정현정 한국조리과학회 1997 한국식품조리과학회지 Vol.13 No.2

본 연구에서는 식품내의 ethyl carbamate 전구체와 그 형성에 영향을 주는 인자들을 규명하고자 하였다. 일반적으로 제조조건 보다는 원료 메주가 간장의 ethyl carbamate 형성에 기여도가 크나, 같은 메주로 제조한 간장의 경우, 저장 기간에 따라 생성이 증가하며 빛에 의해 그 농도가 감소하는 것이 관찰되었다. 따라서, 가정에서 간장을 담을 경우 전통적인 방법인 빛을 쪼여 주며 보관하는 주의가 필요하다고 사료된다. 원료 메주에 따른 차이는 원료 콩의 화학적 조성도 관여를 하겠으나, 발효에 관여하는 미생물의 종류와 그에 따른 대사과정, 대사량의 차이에 의한 영향도 무시할 수 없으리라 사료된다. 따라서 이러한 기전들을 밝혀내기 위하여 앞으로 연구가 계속 진행되어야 할 것으로 사료된다. Ethyl carbamate is an animal carcinogen and a by-product of fermentation. Since the highest level of ethyl carbamate was found in a traditional soy sauce sample in the previous study, soysauce was prepared in varying experimental conditions with three kinds of meju originated from different area of the country to investigate the factors affecting ethyl carbamate formation. The sample analysis methods included a semi-purification with solid-phase extraction and detection by GC-MS at SIM mode using m/z 62 fragmention. Among the investigated factors, which includes source meju, brining length, salt content, irradiation of visible light during brining or storage, and storage length, source meju and storage length showed most profound effect on the formation of ethyl carbamate. Irradiation of visible light for 12 hours a day during storage lowered the concentration of ethyl carbamate compared to the soysauce kept in the dark. Meanwhile irradiation during the brining as well as the degree of salt contents showed no effect.

한국 전통 발효식품 중의 Ethyl Carbamate 정량

정현정,권훈정 동아시아식생활학회 1997 동아시아식생활학회지 Vol.7 No.1

Diet is generally accepted as one of the important factors in human cancer development. Ethyl carbarmate has been associated with cancer for several decades and mainly found in the fermented beverages and foodstuff. The relationship between ethyl carbarmate and the human health cannot be ignored especially in the areas where fermented foods consists of regular food consumption. To investigate the ethyl carbamate exposure level in Korean population, commercial fermented food samples were collected from local markets in Seoul area and home-made varieties were collected throughout the country. Following partial purification, the concentration of ethyl carbamate was determined by GC/MS. The concentration of ethyl carbamate ranged to 70 ppb in soysauce, to 10 ppb in soybean paste, and to 5 ppb in vinegars. Korean traditional alcoholic beverages showed small amount of ethyl carbarmate. The estimated daily exposure of Korean population was 0~1900ng/day. It would be prudent to put efforts to minimize the formation of ethyl carbamate since the risk from the higher range exposure cannot be ignored.