| Summary: | 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.
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