title: Striking Effects of Storage Buffers on Apparent Half-Lives of the Activity of Pseudomonas aeruginosa Arylsulfatase.
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 (t 0.5) at 37 °C were tested. At pH 7.4, PAAS showed non-exponential decreases of activity, with the apparent t 0.5 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 t 0.5 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.
authors.affiliation: Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Yuwei
authors.initials: Y
authors.lastname: Li
authors.affiliation: Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Yang
authors.affiliation: Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Deqiang
authors.initials: D
authors.lastname: Wang
authors.affiliation: Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Hu
authors.affiliation: Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Yuan
authors.affiliation: Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Pu
authors.affiliation: Department of Pharmaceutical Sciences, Molecular Modeling and Biopharmaceutical Center, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536, USA.
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
authors.affiliation: Key Lab of Antibiotics Biotechnology of the Health Ministry, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China.
authors.forename: Zhaoyong
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China. liaofeish@yeah.net.
authors.forename: Fei
authors.initials: F
authors.lastname: Liao
mesh_terms: D001192:Arylsulfatases / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001881:Borates / Q000737:chemistry*; D002021:Buffers; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006207:Half-Life; D007700:Kinetics; D009578:Nitrobenzenes / Q000737:chemistry; D059748:Proteolysis; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D001881:Borates; D002021:Buffers; D009578:Nitrobenzenes; D011994:Recombinant Proteins; C023379:4-nitrophenyl sulfate; C010634:sodium borate; D001192:Arylsulfatases
keywords: Borate; Contaminated proteases; Degradation; Pseudomonas aeruginosa arylsulfatase; Thermostability