Isolation and Characterization of Two Isoperoxidases from Mung Bean Seedling

녹두(綠豆)에서 Peroxidase 동위(同位) 효소(酵素)들의 분리(分離)와 효소적(酵素的) 특성(特性)

The changes in peroxidase activity and its isozyme pattern in the different parts of mung bean sprout were investigated; The enzyme activity in cotyledon and root showed a tendency to increase at an early stage and then decreased gradually as germination continued. However, the crude homogenate of epicotyl and hypocotyl showed a continuous decline in the enzyme activity. In particular, the enzyme activity of the root was $1.5{\sim}3.5$ times higher than that of other parts. Gel electrophoresis of the crude homogenate revealed that the number of isozyme in every part of the mung bean sprout increase during germination up to 6th days. Two isozymes from root were partially purified by ammonium sulfate fractionation, gel filtration by Sephadex G-75 and DEAE cellulose column chromatography. One of the isozymes (A) was purified 16-fold by the present procedure, but the purity of the other isosyme (B) was not increased , significantly. Isozyme A was the most active at $65^{\circ}C$ and isozyme B at $70^{\circ}C$, while both isozyme (A, B) have a optimal pH of 5.6. The Km values of isozyme A and B for 0-dianisidine as a hydrogen donor determined to be 0.071 mM and 0.052mM, respectively, and those for isozyme A and B using $H_2O_2$ as a hydrogen acceptor were 0.28mM and 0.23mM, respectively.

발아초기(發芽初期) 녹두(綠豆)의 부위별(部位烈) peroxidase 활성(活性)은 뿌리가 타부위(他部位)보다 약 $1.5{\sim}3.5$배 더 높은 효소활성(酵素活性)을 나타내었으며 발아(發芽)가 진행(進行)됨에 따라 자엽(予葉), 뿌리에서는 효소활성(酵素活性)이 증가(增加)하다가 점차 감소(滅少)하는 경향(傾向)을 보인 반면에 타부위(他部位)에서는 계속 감소(減少)하는 경향(傾向)을 보였다. 전기영동상(電氣泳動上) isozyme의 수는 발아(發芽), 생육(生育)이 진행(進行)됨에 따라 모든 부위(部位)에서 계속 증가(增加)하여 활성도(活性度)와는 무관(無關)하게 발아(發芽) 후 6일(日)째에 가장 많이 나타났다. Ammonium sulfate에 의한 단백질(蛋白質)의 분획(分劃), Sephadex G-75 Column, DEAE-cellulose column한 결과(結果) isozyme A는 16배 정제(精製)되었으며, isozyme B는 조금도 순도(純度)가 증가(增加)되지 않았으며 isozyme A와 B의 전기영동상(電氣泳動上) Rm값은 각각(各各) 0.22와 0.62였다. isozyme A와 B 모두 PH5.6에서 최대(最大) 활성(活性)을 나타내었으며 또한 반응최적온도(反應最適溫度)는 각각(各各) $65^{\circ}C$와 $70^{\circ}C$였으며, 열(熱)에 대해서도 비교적(比較的) 안정(安定)한 결과(結果)를 얻었다. Lineweaver-Burk Polt를 이용(利用)하며 Km값을 측정(測定)해 본 결과(結果) 0-dianisidine에 대한 isozyme A는 0.071mM, isozyme B는 0.052mM이었으며 $H_2O_2$에 대한 isozyme A는 0.28mM, isozyme B는 0.23mM이었다.