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- 저자 : 김승수 ( Han Seung Joo (검색결과 5 건)
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Inactivation of the Peroxidase Activity of Rabbit Hemoglobin by Diethylpyrocarbonate
주한승,김승수,Joo, Han-Seung,Kim, Soung-Soo 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.6
토끼 헤모글로빈의 peroxidase 활성은 histidine에 비교적 특이하게 반응하는 diethylpyrocarbonate에 의하여 불활성화 되었다. 50 mM sodium phosphate 완충용액 (pH 6.0)을 사용하였을 때 $30^{\circ}C$에서 modification되는 속도상수는 $220M^{-1}min^{-1}$이었으며, N-carbethoxylhistidyl 유도체의 생성으로 인한 242 nm에서의 흡광도 증가가 동시에 관찰되었다. 그러나 280nm에서의 흡광도 변화는 관찰되지 않았으며 토끼 헤모글로빈의 peroxidase 활성은 N-acetylimidazole, phenylglyoxal 및 N-ethylmaleimide 등에 의하여 활성이 저하되지 않았다. Hydroxylamine은 peroxidase 활성의 inhibitor로 작용하기 때문에 diethylpyrocarbonate에 의하여 불활성화된 토끼 헤모글로빈의 peroxidase 활성을 회복시키지 못했지만, 기질인 guaiacol은 diethylpyrocabonate에 의한 불활성화를 약간 감소시켰다. Tsou의 통계적 분석방법에 의하여 peroxidase 활성이 완전히 사라질 때 헤모글로빈 1분자당 11개의 histidine 잔기가 modification되었으며 그 중 1개의 histidine만이 peroxidase 활성에 필수적인 것으로 밝혀졌다. 또한 5 mM $H_2O_2$로 처리한 토끼 헤모글로빈의 경우 diethylpyrocarbonate에 의한 불활성화가 촉진되고, diethylpyrocarbonate로 modification된 apohemoglobin이 native 헤모글로빈으로 재조합하지 못하는 것으로 보아 essential histidine이 heme 근처의 distal histidine일 것으로 추정된다. Diethylpyrocarbonate inhibits the peroxidase activity of rabbit hemoglobin with a second order rate constant of $220M^{-1}min^{-1}$ at pH 6.0 and $30^{\circ}C$. The increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives and other modification studies suggest that the modification of histidine residues is responsible for the loss of activity. However, treatment of inactivated hemoglobin with hydroxylamine does not restore catalytic activity due to the inhibitory effect of hydroxylamine itself on the peroxidase activity of hemoglobin. The substrate guaiacol have partially reduced the diethylpyrocarbonate dependent inactivation, and a reconstitution experiment using diethylpyrocarbonate treated apohemoglobin shows that it is not reconstituted to hemoglobin having full activity. The decrease of absorption at Soret region (406 nm) upon diethylpyrocarbonate modification of native hemoglobin and the acceleration of inactivation by preincubation of the diethylpyrocarbonate inactivated hemoglobin with $H_2O_2$ (5 mM) implies that the essential residue is distal histidine as previously proposed in case of cytochrome C peroxidase.
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Diethylpyrocarbonate 에 의한 토끼 헤모글로빈의 Peroxidase 활성의 불활성화
주한승,김승수 ( Han Seung Joo,Soung Soo Kim ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.6
Diethylpyrocarbonate inhibits the peroxidase activity of rabbit hemoglobin with a second order rate constant of 220 M^(-1)min^(-1) at pH 6.0 and 30℃. The increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives and other modification studies suggest that the modification of histidine residues is responsible for the loss of activity. However, treatment of inactivated hemoglobin with hydroxylamine does not restore catalytic activity due to the inhibitory effect of hydroxylamine itself on the peroxidase activity of hemoglobin. The substrate guaiacol have partially reduced the diethylpyrocarbonate dependent inactivation, and a reconstitution experiment using diethylpyrocarbonate treated apohemoglobin shows that it is not reconstituted to hemoglobin having full activity. The decrease of absorption at Soret region (406 nm) upon diethylpyrocarbonate modification of native hemoglobin and the acceleration of inactivation by preincubation of the diethylpyrocarbonate inactivated hemoglobin with H₂O₂ (5 mM) implies that the essential residue is distal histidine as previously proposed in case of cytochrome C peroxidase.
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토끼 Hemoglobin 의 Peroxidase 활성에 관한 연구
주한승,임주원,김승수 ( Han Seung Joo,Joo Won Lim,Soung Soo Kim ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.6
Rabbit hemoglobin was purified by using ammonium sulfate fractionation, CM-Sephadex chromatography, hydroxyapatite chromatography and Sephadex G-100 gel filtration. The purified hemoglobin oxidized various synthetic and naturally occurring phenolic compounds in the presence of H₂O₂. The substrate specificity studies indicate that rabbit hemoglobin has about 10 times lower K_m values for guaiacol (0.39 mM) and o-dianisidine (0.09 mM) when compared to those of plant peroxidases. Among natural substrates tested, rabbit hemoglobin catalyzes the oxidation of caffeic acid, chlorogenic acid and indole-3-acetic acid quite rapidly compare to other naturally occurring phenolic compounds such as scopoletin, esculetin, ferulic acid and p-coumaric acid. In general, phenolic compounds having methoxy group appears to have high affinity to rabbit hemoglobin as revealed by their very low K_m values. Chemical modification studies indicate that p-chloromercuribenzoate inactivates the peroxidase activity of rabbit hemoglobin, whereas N-ethylmaleimide or iodoacetamide does not. Furthermore, the peroxidase activity of rabbit hemoglobin is rapidly inactivated by diethylpyrocarbonate, a histidyl selective reagent, unlike plant peroxidases.
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Studies on the Peroxidase Activity of Rabbit Hemoglobin
주한승,임주원,김승수,Joo, Han-Seung,Lim, Joo-Won,Kim, Soung-Soo 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.6
토끼 erythrocyte lysate으로부터 hemoglobin을 $(NH_4)_2SO_4$ 분별침전, CM-Sephadex 크로마토그래피, hydroxyapatite 크로마토그래피, Sephadex G-l00 겔 크로마토그래피 방법을 이용하여 순수 정제하였다. 정제한 토끼 hemoglobin은 peroxidase 활성을 나타냈으며 guaiacol과 o-dianisidine을 기질로 사용하였을 경우 식물 peroxidase보다 10배 가량 낮은$K_m$값을 나타냈다. 여러 phenol 화합물들을 기질로 사용하였을 때 토끼 hemoglobin은 caffeic acid, chlorogenic acid, indole-3-acetic acid를 빠르게 산화하였으며, disubstituted phenol 화합물과 methoxylated phenol 화합물들이 rabbit hemoglobin에 대하여 친화력이 큰 것으로 나타났다. Chemical modification 실험결과 토끼 hemoglobin은 iodoacetamide와 N-ethylmaleimide에 의하여 활성이 억제되지 않지만 p-chloromercuribenzoate에 의해서는 활성이 현저히 억제되었다. 또한 식물 peroxidase와는 달리 hemoglobin의 peroxidase 활성은 diethylpyrocarbonate에 의하여 급격히 불활성화 되었다. Rabbit hemoglobin was purified by using ammonium sulfate fractionation, CM-Sephadex chromatography, hydroxyapatite chromatography and Sephadex G-100 gel filtration. The purified hemoglobin oxidized various synthetic and naturally occurring phenolic compounds in the presence of $H_2O_2$. The substrate specificity studies indicate that rabbit hemoglobin has about 10 times lower $K_m$ values for guaiacol (0.39 mM) and o-dianisidine (0.09 mM) when compared to those of plant peroxidases. Among natural substrates tested, rabbit hemoglobin catalyzes the oxidation of caffeic acid, chlorogenic acid and indole-3-acetic acid quite rapidly compare to other naturally occurring phenolic compounds such as scopoletin, esculetin, ferulic acid and p-coumaric acid. In general, phenolic compounds having methoxy group appears to have high affinity to rabbit hemoglobin as revealed by their very low $K_m$ values. Chemical modification studies indicate that p-chloromercuribenzoate inactivates the peroxidase activity of rabbit hemoglobin, whereas N-ethylmaleimide or iodoacetamide does not. Furthermore, the peroxidase activity of rabbit hemoglobin is rapidly inactivated by diethylpyrocarbonate, a histidyl selective reagent, unlike plant peroxidases.
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