Journal Article

States of Tryptophyl Residues and Stability of Recombinant Human Matrix Metalloproteinase 7 (Matrilysin) as Examined by Fluorescence

Kuniyo Inouye, Hiromi Tanaka and Hiroshi Oneda

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 128, issue 3, pages 363-369
Published in print September 2000 | ISSN: 0021-924X
Published online September 2000 | e-ISSN: 1756-2651 | DOI: http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022762
States of Tryptophyl Residues and Stability of Recombinant Human Matrix Metalloproteinase 7 (Matrilysin) as Examined by Fluorescence

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States of tryptophyl residues and stability of human matrilysin were studied. The activation energy for the thermal inactivation of matrilysin was determined to be 237kJ/mol, and 50% of the activity was lost upon incubation at 69°C for 10 min. The activity was increased by adding Nacl, and was doubled with 3 M Nacl. Denaturation of matrilysin by guanidine hydrochloride (GdnHCl) and urea was monitored by fluorescence change of tryptophyl residues. Half of the change was observed at 2.2-2.7 M GdnHCl, whereas no change was observed even with 8 M urea. Half of the inactivation was induced at 0.8 M GndHCl and at 2 M urea. The presence of an inactive intermediate with the same fluorescence spectrum as the native enzyme was suggested in the denatureation. Matrilysin contains for tryptophyls, and their states were examined by fluorescence-quenching with iodide and cesium ions and acrylamide. No tryptophyls in the native enzyme were accessible to I and Cs+, and 2.4 residues were accessible to acrylamide. Based on the crystallographic study, Trp 154 is water-accessible, but it should be in a crevice not to contact with I and Cs+. All tryptophyls in the GdnHCl-denatured enzyme were exposed to the quenchers, while a considerable part was inaccessible in the urea-denatured one

Keywords: denaturation; fluorescence quenching; matrilysin; matrix metalloproteinase; protein stability

Journal Article.  0 words. 

Subjects: Biochemistry

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