Striking Effects of Storage Buffers on Apparent Half-Lives of the Activity of Pseudomonas aeruginosa Arylsulfatase

Yuwei Li, Xiaolan Yang, Deqiang Wang, Xiaolei Hu, Mei Yuan, Jun Pu, Chang Guo Zhan, Zhaoyong Yang, Fei Liao

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

To obtain the label enzyme for enzyme-linked-immunoabsorbent-assay of two components each time in one well with conventional microplate readers, molecular engineering of Pseudomonas aeruginosa arylsulfatase (PAAS) is needed. To compare thermostability of PAAS/mutants of limited purity, effects of buffers on the half-activity time (t0.5) at 37 °C were tested. At pH 7.4, PAAS showed non-exponential decreases of activity, with the apparent t0.5 of ~6.0 days in 50 mM HEPES, but ~42 days in 10 mM sodium borate with >85 % activity after 15 days; protein concentrations in both buffers decreased at slower rates after there were significant decreases of activities. Additionally, the apparent t0.5 of PAAS was ~14 days in 50 mM Tris–HCl, and ~21 days in 10 mM sodium phosphate. By sodium dodecyl-polyacrylamide gel electrophoresis, the purified PAAS gave single polypeptide; after storage for 14 days at 37 °C, there were many soluble and insoluble fragmented polypeptides in the HEPES buffer, but just one principal insoluble while negligible soluble fragmented polypeptides in the borate buffer. Of tested mutants in the neutral borate buffer, rates for activity decreases and polypeptide degradation were slower than in the HEPES buffer. Hence, dilute neutral borate buffers were favorable for examining thermostability of PAAS/mutants.

Original languageEnglish
Pages (from-to)283-290
Number of pages8
JournalProtein Journal
Volume35
Issue number4
DOIs
StatePublished - Aug 1 2016

Bibliographical note

Publisher Copyright:
© 2016, Springer Science+Business Media New York.

Keywords

  • Borate
  • Contaminated proteases
  • Degradation
  • Pseudomonas aeruginosa arylsulfatase
  • Thermostability

ASJC Scopus subject areas

  • Analytical Chemistry
  • Bioengineering
  • Biochemistry
  • Organic Chemistry

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