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Proteinase K activity determination with β-galactosidase as sensitive macromolecular substrate
- Source :
- Analytical Biochemistry. 513:54-60
- Publication Year :
- 2016
- Publisher :
- Elsevier BV, 2016.
-
Abstract
- Proteinase K from Engyodontium album (proK) is a relatively unspecific serine endopeptidase which is known to attack proteins yet in their native states. If the attacked protein is an enzyme, even a partial hydrolysis by proK may lead to an inactivation of the enzyme, which can be monitored by measuring the loss of catalytic activity of the attacked enzyme. E. coli β-galactosidase (β-Gal) was used in this work as such enzyme. It was found to be a convenient and sensitive macromolecular model substrate for comparing the “native protein-attacking ability” of free and immobilized proK at pH = 7.0 and 23 °C. The β-Gal activity was measured spectrophotometrically with o -nitrophenyl-β-galactopyranoside. Reproducible proK determinations were possible for as little as 4.3 ng proK by using a proK analyte solution of 10 nM. Compared to free proK, immobilized proK was much less efficient in inactivating β-Gal, most likely due to a decreased mobility of immobilized proK and a restricted accessibility of β-Gal to the active site of proK. Worth noting is, that under conditions at which β-Gal was completely inactivated by proK, the activity of hen egg lysozyme, horseradish peroxidase, or Aspergillus sp. glucose oxidase remained unaltered.
- Subjects :
- Immobilized enzyme
Biophysics
02 engineering and technology
01 natural sciences
Biochemistry
Horseradish peroxidase
Fungal Proteins
Ascomycota
Escherichia coli
Glucose oxidase
Molecular Biology
chemistry.chemical_classification
biology
Escherichia coli Proteins
010401 analytical chemistry
Proteolytic enzymes
Active site
Substrate (chemistry)
Cell Biology
Enzymes, Immobilized
beta-Galactosidase
021001 nanoscience & nanotechnology
Proteinase K
Molecular biology
0104 chemical sciences
Enzyme
chemistry
biology.protein
Endopeptidase K
0210 nano-technology
Subjects
Details
- ISSN :
- 00032697
- Volume :
- 513
- Database :
- OpenAIRE
- Journal :
- Analytical Biochemistry
- Accession number :
- edsair.doi.dedup.....c1e0e40e57da66fa9f6c5114444c9a55