Thermal inactivation of shrimp deoxyribonuclease with and without sodium dodecyl sulfate.
Due to the differences in sample treatment, purified shrimp DNase migrates to different positions in sodium dodecyl sulfate (NaDod-SO4) polyacrylamide gel electrophoresis. DNase molecules migrating to some of these positions are enzymatically active as revealed by the DNase activity stain in situ. When the sample is not heated, DNase molecules (Form I) migrate to a position corresponding to an apparent Mr of 22 000 and are stain DNase-active. When the sample is heated with NaDod-SO4, DNase molecules (Form II) migrate to the apparent Mr of 39 000 position and are also DNase-active. In contrast, when the sample is heated without NaDod-SO4, DNase molecules (Form II') migrate to the same position as Form II but are DNase-inactive. When the sample is heated in the presence of beta-mercaptoethanol (with or without NaDod-SO4), the inactive Form III is generated. Form III must be the fully extended polypeptide because its apparent Mr is very close to the true Mr of DNase. These treated samples were also analyzed for DNase activity by diluting DNase-NaDod-SO4 into the DNase (hyperchromicity) assay solution. The results are consistent in that only Forms I and II are DNase-active. However, Form I differs from Form II in the time required for expression of DNase activity. After dilution of NaDod-SO4. Form II requires approx. 60 min to attain full activity while Form I is active immediately. Form II itself is inactive. The activity found at the Mr 39 000 position is due to conversion of Form II to the active Form I, as revealed by two-dimensional NaDod-SO4 gel electrophoresis. The sample heated without NaDod-SO4 soon contains active Form II, indicating that Form II is an intermediate during the production of Form II'. When Form II' is heated with NaDod-SO4, it does not refold back to the native state through Form II, suggesting that Form II' is irreversibly denatured. Thus, the anomalous behaviors of shrimp DNase in NaDod-SO4 gel electrophoresis have been utilized to study, in the presence and absence of NaDod-SO4, the thermal unfolding, as well as the refolding to the active enzyme during cooling and NaDod-SO4 removal.
Lin
J L
JL
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, R.O.C.
Wang
W Y
WY
Liao
T H
TH
D000818:Animals; D033364:Decapoda; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006358:Hot Temperature; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate
D012967:Sodium Dodecyl Sulfate; D003851:Deoxyribonucleases