Purification procedure and assay for the activity of lysyl oxidase
The goal of the present study was to extract and purify lysyl oxidase from rodent’s tissues by a fast, simple, effective and inexpensive method and to develop a sensitive, time-saving lysyl oxidase specific activity assay for routine in vitro experiments. Lysyl oxidase was purified by elaborated pur...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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National Academy of Sciences of Ukraine, Palladin Institute of Biochemistry
2018-11-01
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| Series: | The Ukrainian Biochemical Journal |
| Subjects: |
| _version_ | 1797641863514030080 |
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| author | .O. O. Gudkova N. V. Latyshko O. V. Zaitseva S. G. Shandrenko |
| author_facet | .O. O. Gudkova N. V. Latyshko O. V. Zaitseva S. G. Shandrenko |
| author_sort | .O. O. Gudkova |
| collection | DOAJ |
| description | The goal of the present study was to extract and purify lysyl oxidase from rodent’s tissues by a fast, simple, effective and inexpensive method and to develop a sensitive, time-saving lysyl oxidase specific activity assay for routine in vitro experiments. Lysyl oxidase was purified by elaborated purification procedure which relies on negative adsorption principle, that is, an effective decrease in the concentration of ballast components by the polar hydrophilic adsorbent and increasing the concentration of the protein of interest. Peroxide-coupled lysyl oxidase activity quantification methods based on luminol chemiluminescence in the presence of horseradish peroxidase as a catalyst and fluorescent detection using folic acid and Cu(II) with 1,5-diaminopentane as the substrate, were designed. Lysyl oxidase was partially purified from urea extracts of rodent’s tissues. Used purification procedure ensures the fast release of 93% of ballast proteins as shown by polyacrylamide gel electrophoresis. Lysyl oxidase specific activity after purification was 10-22-fold higher than that of the original extract. The molecular mass of murine lysyl oxidase from lung and heart was estimated to be ~32 kDa.
We elaborated two sensitive methods for lysyl oxidase activity quantification and fast inexpensive procedure for partial enzyme purification useful in bulky in vitro experiments. |
| first_indexed | 2024-03-11T13:51:45Z |
| format | Article |
| id | doaj.art-f205908d03ed425f8a0a6590c69bc313 |
| institution | Directory Open Access Journal |
| issn | 2409-4943 2413-5003 |
| language | English |
| last_indexed | 2024-03-11T13:51:45Z |
| publishDate | 2018-11-01 |
| publisher | National Academy of Sciences of Ukraine, Palladin Institute of Biochemistry |
| record_format | Article |
| series | The Ukrainian Biochemical Journal |
| spelling | doaj.art-f205908d03ed425f8a0a6590c69bc3132023-11-02T09:03:56ZengNational Academy of Sciences of Ukraine, Palladin Institute of BiochemistryThe Ukrainian Biochemical Journal2409-49432413-50032018-11-019059810510.15407/ubj90.05.098Purification procedure and assay for the activity of lysyl oxidase.O. O. Gudkova0N. V. Latyshko1O. V. Zaitseva2S. G. Shandrenko3Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, KyivPalladin Institute of Biochemistry, National Academy of Sciences of Ukraine, KyivPalladin Institute of Biochemistry, National Academy of Sciences of Ukraine, KyivPalladin Institute of Biochemistry, National Academy of Sciences of Ukraine, KyivThe goal of the present study was to extract and purify lysyl oxidase from rodent’s tissues by a fast, simple, effective and inexpensive method and to develop a sensitive, time-saving lysyl oxidase specific activity assay for routine in vitro experiments. Lysyl oxidase was purified by elaborated purification procedure which relies on negative adsorption principle, that is, an effective decrease in the concentration of ballast components by the polar hydrophilic adsorbent and increasing the concentration of the protein of interest. Peroxide-coupled lysyl oxidase activity quantification methods based on luminol chemiluminescence in the presence of horseradish peroxidase as a catalyst and fluorescent detection using folic acid and Cu(II) with 1,5-diaminopentane as the substrate, were designed. Lysyl oxidase was partially purified from urea extracts of rodent’s tissues. Used purification procedure ensures the fast release of 93% of ballast proteins as shown by polyacrylamide gel electrophoresis. Lysyl oxidase specific activity after purification was 10-22-fold higher than that of the original extract. The molecular mass of murine lysyl oxidase from lung and heart was estimated to be ~32 kDa. We elaborated two sensitive methods for lysyl oxidase activity quantification and fast inexpensive procedure for partial enzyme purification useful in bulky in vitro experiments.1-5-diaminopentanechemiluminescent methoddensitogramfluorometric assaykaolinitelysyl oxidasenegative adsorptionpolyacrylamide gel |
| spellingShingle | .O. O. Gudkova N. V. Latyshko O. V. Zaitseva S. G. Shandrenko Purification procedure and assay for the activity of lysyl oxidase The Ukrainian Biochemical Journal 1-5-diaminopentane chemiluminescent method densitogram fluorometric assay kaolinite lysyl oxidase negative adsorption polyacrylamide gel |
| title | Purification procedure and assay for the activity of lysyl oxidase |
| title_full | Purification procedure and assay for the activity of lysyl oxidase |
| title_fullStr | Purification procedure and assay for the activity of lysyl oxidase |
| title_full_unstemmed | Purification procedure and assay for the activity of lysyl oxidase |
| title_short | Purification procedure and assay for the activity of lysyl oxidase |
| title_sort | purification procedure and assay for the activity of lysyl oxidase |
| topic | 1-5-diaminopentane chemiluminescent method densitogram fluorometric assay kaolinite lysyl oxidase negative adsorption polyacrylamide gel |
| work_keys_str_mv | AT oogudkova purificationprocedureandassayfortheactivityoflysyloxidase AT nvlatyshko purificationprocedureandassayfortheactivityoflysyloxidase AT ovzaitseva purificationprocedureandassayfortheactivityoflysyloxidase AT sgshandrenko purificationprocedureandassayfortheactivityoflysyloxidase |