Study into the Kinetic Properties and Surface Attachment of a Thermostable Adenylate Kinase

Abstract: A thermostable adenylate kinase (tAK) has been used as model protein contaminant on surfaces, so used because residual protein after high temperature wash steps can be detected at extremely low concentrations. This gives the potential for accurate, quantitative measurement of the effectiveness of different wash processes in removing protein contamination. Current methods utilise non-covalent (physisorbtion) of tAK to surfaces, but this can be relatively easily removed. In this study, the covalent binding of tAK to surfaces was studied to provide an alternative model for surface contamination. Kinetic analysis showed that the efficiency of the enzyme expressed as the catalytic rate over the Michaelis constant (kcat/KM) increased from 8.45 ± 3.04 mM-1 s-1 in solution to 32.23 ± 3.20 or 24.46 ± 4.41 mM-1 s-1 when the enzyme was immobilised onto polypropylene or plasma activated polypropylene respectively. Maleic anhydride plasma activated polypropylene showed potential to provide a more robust challenge for washing processes as it retained significantly higher amounts of tAK enzyme than polypropylene in simple washing experiments. Inhibition of the coupled enzyme (luciferase/luciferin) system used for the detection of adenylate kinase activity, was observed for a secondary product of the reaction. This needs to be taken into consideration when using the assay to estimate cleaning efficacy.

Subjects:
Biomolecules and biochemistry
Catalysis and surfaces
Microbial sciences

Cite this dataset as:
Hathaway, H., 2017. Study into the Kinetic Properties and Surface Attachment of a Thermostable Adenylate Kinase. Bath: University of Bath Research Data Archive. Available from: https://doi.org/10.15125/BATH-00334.

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Data

0.1mM_ADP.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (37kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface and light output measured as luminescence generated as a function of ATP production utilising 0.1mM ADP as substrate. File includes calculation of initial rates and standard deviation. Also includes washing experiment data for 40ng tAK.

0.05mM_ADP_PP.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (31kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface and light output measured as luminescence generated as a function of ATP production utilising 0.05mM ADP as substrate. File includes calculation of initial rates and standard deviation.

0.0016.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (29kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface and light output measured as luminescence generated as a function of ATP production utilising 0.0016mM ADP as substrate. File includes calculation of initial rates and standard deviation.

0.025.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (30kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface and light output measured as luminescence generated as a function of ATP production utilising 0.025mM ADP as substrate. File includes calculation of initial rates and standard deviation.

0.0031.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (29kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface and light output measured as luminescence generated as a function of ATP production utilising 0.0031mM ADP as substrate. File includes calculation of initial rates and standard deviation.

0.0063.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (30kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface and light output measured as luminescence generated as a function of ATP production utilising 0.0063mM ADP as substrate. File includes calculation of initial rates and standard deviation.

0.0125.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (30kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface and light output measured as luminescence generated as a function of ATP production utilising 0.0125mM ADP as substrate. File includes calculation of initial rates and standard deviation.

0mM.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (28kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface and light output measured as luminescence generated as a function of ATP production utilising 0mM ADP as substrate. File includes calculation of initial rates and standard deviation.

0.1mM.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (38kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface activated by plasma deposition of maleic anhydride and light output measured as luminescence generated as a function of ATP production utilising 0.1mM ADP as substrate. File includes calculation of initial rates and standard deviation. Also includes washing experiment data for 40ng tAK.

0.05mM_ADP_MAPP.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (30kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface activated by plasma deposition of maleic anhydride and light output measured as luminescence generated as a function of ATP production utilising 0.05mM ADP as substrate. File includes calculation of initial rates and standard deviation.

0.0016mM_ADP.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (29kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface activated by plasma deposition of maleic anhydride and light output measured as luminescence generated as a function of ATP production utilising 0.0016mM ADP as substrate. File includes calculation of initial rates and standard deviation.

0.025mM_ADP.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (31kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface activated by plasma deposition of maleic anhydride and light output measured as luminescence generated as a function of ATP production utilising 0.025mM ADP as substrate. File includes calculation of initial rates and standard deviation.

0.0031mM_ADP.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (30kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface activated by plasma deposition of maleic anhydride and light output measured as luminescence generated as a function of ATP production utilising 0.0031mM ADP as substrate. File includes calculation of initial rates and standard deviation.

0.0063mM_ADP.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (30kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface activated by plasma deposition of maleic anhydride and light output measured as luminescence generated as a function of ATP production utilising 0.0063mM ADP as substrate. File includes calculation of initial rates and standard deviation.

0.0125mM_ADP.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (30kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface activated by plasma deposition of maleic anhydride and light output measured as luminescence generated as a function of ATP production utilising 0.0125mM ADP as substrate. File includes calculation of initial rates and standard deviation.

0mM_ADP.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (26kB)

Raw data showing time course experiment of tAK adsorbed to a polypropylene surface activated by plasma deposition of maleic anhydride and light output measured as luminescence generated as a function of ATP production utilising 0mM ADP as substrate. File includes calculation of initial rates and standard deviation.

Initial_rates_1ng_tAK.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (35kB)

Solution data for tAK (1ng) as a function of ADP concentration used to calculate initial rates in solution

Initial_rates_5ng_tAK.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (34kB)

Solution data for tAK (5ng) as a function of ADP concentration used to calculate initial rates in solution

Initial_rates_10ng_tAK.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (38kB)

Solution data for tAK (10ng) as a function of ADP concentration used to calculate initial rates in solution

Initial_rates_20ng_tAK.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (39kB)

Solution data for tAK (20ng) as a function of ADP concentration used to calculate initial rates in solution

Initial_rates_40ng_tAK.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (38kB)

Solution data for tAK (40ng) as a function of ADP concentration used to calculate initial rates in solution

Initial_Rates_-6-_-9.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (26kB)

Initial rate of light production raw data as a function of ATP concentration (10^-6 to 10^-9M ATP) used to generate standard curve

Initial_Rates_10-14.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (18kB)

Initial rate of light production raw data as a function of ATP concentration (10^-10 to 10^-14M ATP) used to generate standard curve

Standard_Curve … Endpoints.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (27kB)

Raw data showing initial rate of light production as a function of ATP concentration including averages and standard deviation

50ul_luc., … ATP,_Vary_AMP.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (26kB)

Raw data showing effect of AMP on luciferase turnover of ATP

IR-_PP-STRIP_+_PP-MA-STRIP.xlsx
application/vnd.openxmlformats-officedocument.spreadsheetml.sheet (164kB)

IR spectra raw data showing polypropylene with and without maleic anhydride deposition

Creators

Hollie Hathaway
University of Bath

Contributors

Toby Jenkins
Supervisor
University of Bath

0000-0002-8981-3029

Mark Sutton
Supervisor
Public Health England

University of Bath
Rights Holder

Coverage

Collection date(s):

From 2014 to 2015

Documentation

Data collection method:

UV-Vis Luminescence spectroscopy. IR spectroscopy.

Data processing and preparation activities:

Data worked up in Microsoft Excel and presented in Origin Graphing Software.

Methodology link:

Hathaway, H.J., Sutton, J.M., and Jenkins, A.T.A., 2015. Study into the kinetic properties and surface attachment of a thermostable adenylate kinase. Biochemistry and Biophysics Reports, 1, 1-7. Available from: https://doi.org/10.1016/j.bbrep.2015.03.011.

Funders

Biotechnology and Biological Sciences Research Council (BBSRC)
https://doi.org/10.13039/501100000268

iCase PhD Studentship

Public Health England (PHE)
https://doi.org/10.13039/501100002141

Publication details

Publication date: 2017
by: University of Bath

Version: 1

DOI: https://doi.org/10.15125/BATH-00334

URL for this record: https://researchdata.bath.ac.uk/id/eprint/334

Related papers and books

Hathaway, H.J., Sutton, J.M., and Jenkins, A.T.A., 2015. Study into the kinetic properties and surface attachment of a thermostable adenylate kinase. Biochemistry and Biophysics Reports, 1, 1-7. Available from: https://doi.org/10.1016/j.bbrep.2015.03.011.

Contact information

Please contact the Research Data Service in the first instance for all matters concerning this item.

Contact person: Hollie Hathaway

Departments:

Faculty of Science
Chemistry

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