Chicken Pancreatic Deoxyribonuclease:Effects of Calcium Ion on the Enzymatic Activity and the Opening and Reformation of Disulfide Bonds

• Main Authors: Cheng, chaw-chen, 鄭兆珍
• Other Authors: Ming-Fu Chang
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/38393193535770884089

碩士 === 國立臺灣大學 === 生化學研究所 === 85 === Abstract The method previously established in our laboratory for purification of chicken pancreatic DNase I proved to be useful. However, it suffered fromlow recovery and inconsistency. A simplified and reproducible method wasthus necessary. Here we develop the following steps : DNase I was first precipitated by ammonium sulfate with the addition of acetic acid to bring the pH to 5.0, followed by chromatographic purification using DEAE- cellulose, phenyl-Sepharose, and Hydroxyapatite columns. The resultingenzyme preparation was homogeneous as confirmed by SDS- PAGE. During the purification process, Ca2+ was found to play an important role on the enzymatic activity and stability. The enzyme appeared to be unstable during chromatography using anion-exchangers, such as DEAE-cellulose and HighQ. It is important to properly regulate the flow rate and the concentration gradient profile to shorten the time for sample to flow through the column. The modified method was very useful to purify chicken pancreatic DNase I. There are 4 Cys forming two disulfide bonds in bovine pancreatic DNase I, whereas 6 Cys are in chicken pancreatic DNase I. Based on the 3-dimensionalstructure of bovine pancreatic DNase I, this two extra Cys (Cys-192 and Cys-217) may form an additional disulfide bond. Since Ca2+ has a significant effect on the reduction of disulfide bonds of bovine pancreatic DNase I, it would be interesting to study also this Ca2+ effect on chicken pancreatic DNase I. Using Mn2+-DNA as substrate, Ca2+ increases the activity of bovine pancreatic DNase I by 30 % whereas there is DNase I under the same conditions,a significant 6 fold increase for chicken. In the absence of Ca2+, β-MSH inactivates the activity of bovine pancreatic DNase I to less than 10 %. After addition of Ca2+, the activity goes back to 60 % of its original level.As for chicken pancreatic DNase I, addition of β-MSH leads only to approximately a 30 % decrease in activity, and the activity remained unchanged even after the addition of Ca2+. In order to study the relationship between the degree of disulfide bond opening and the activity of DNase I, the disulfide bonds were reduced, in the presence and absence of Ca2+, by β-MSH followed by alkylation with IAM. The enzyme activity and amino acids composition were then determined to estimate the number of Cys being reduced.