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. 1982 Nov 1;207(2):201–205. doi: 10.1042/bj2070201

Effects of pH and urea on the conformational properties of subtilisin DY.

F Ricchelli, G Jori, B Filippi, R Boteva, M Shopova, N Genov
PMCID: PMC1153849  PMID: 6818946

Abstract

Subtilisin DY is very resistant to the denaturing action of urea: the conformational properties are not affected up to 4.5 M-urea, and even in the presence of 8 M-urea there is only a slow loss of ordered structure and caseinolytic activity. C.d. and fluorescence-emission studies also show that this proteinase is stable in the 5.5-10.0 pH range, whereas below pH 5.5 a sharp denaturation occurs that is complete at pH 4.5. Protein denaturation leads to a change of the emission quantum yield; in particular, in the native protein, indole fluorescence is quenched by some amino groups. Moreover, subtilisin DY possesses two classes of tyrosine residues: one class of exposed residues titrates normally, with pKapp. = 10.24, whereas one class of partially buried or hydrogen-bonded residues ionizes with pKapp. = 11.58. In general, such conformational properties resemble those of other subtilisins. However, some differences occur: e.g., subtilisin DY is less stable at acidic pH values and its tyrosine residues are more accessible to the solvent. Such differences are probably due to small variations of the three-dimensional structure; e.g., subtilisin DY has a slightly lower alpha-helix content.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brown M. F., Omar S., Raubach R. A., Schleich T. Quenching of the tyrosyl and tryptophyl fluorescence of subtilisins Carlsberg and Novo by iodide. Biochemistry. 1977 Mar 8;16(5):987–992. doi: 10.1021/bi00624a028. [DOI] [PubMed] [Google Scholar]
  2. Brown M. F., Schleich T. Circular dichroism and gel filtration behavior of subtilisin enzymes in concentrated solutions of guanidine hydrochloride. Biochemistry. 1975 Jul 15;14(14):3069–3074. doi: 10.1021/bi00685a005. [DOI] [PubMed] [Google Scholar]
  3. Brown M. F., Schleich T. Resolution of independently titrating spectral components in the ultraviolet circular dichroism of subtilisin enzymes by matrix rank analysis. Biochim Biophys Acta. 1977 Nov 23;485(1):37–51. doi: 10.1016/0005-2744(77)90191-7. [DOI] [PubMed] [Google Scholar]
  4. Chen Y. H., Yang J. T., Chau K. H. Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. Biochemistry. 1974 Jul 30;13(16):3350–3359. doi: 10.1021/bi00713a027. [DOI] [PubMed] [Google Scholar]
  5. Edelhoch H., Brand L., Wilchek M. Fluorescence studies with tryptophyl peptides. Biochemistry. 1967 Feb;6(2):547–559. doi: 10.1021/bi00854a024. [DOI] [PubMed] [Google Scholar]
  6. Genov N., Shopova M., Boteva R., Jori G., Ricchelli F. Chemical, photochemical and spectroscopic characterization of an alkaline proteinase from Bacillus subtilis variant DY. Biochem J. 1982 Nov 1;207(2):193–200. doi: 10.1042/bj2070193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Genov N. Spectrophotometric titration of tyrosyl residues in alkaline mesentericopeptidase. Int J Pept Protein Res. 1975;7(4):325–332. doi: 10.1111/j.1399-3011.1975.tb02447.x. [DOI] [PubMed] [Google Scholar]
  8. Gounaris A., Ottesen M. Some properties of succinylated subtilopeptidase. C R Trav Lab Carlsberg. 1965;35(3):37–62. [PubMed] [Google Scholar]
  9. Grossweiner L. I. Application of diffusion theory to photodynamic damage in large targets. Photochem Photobiol. 1977 Sep;26(3):309–311. doi: 10.1111/j.1751-1097.1977.tb07490.x. [DOI] [PubMed] [Google Scholar]
  10. Herskovits T. T., Fuchs H. H. Solvent perturbation of the tyrosyl and tryptophyl residues in subtilisin BPN'. Biochim Biophys Acta. 1972 May 18;263(3):468–476. doi: 10.1016/0005-2795(72)90028-1. [DOI] [PubMed] [Google Scholar]
  11. Markland F. S., Brown D. M., Smith E. L. Subtilisin Amylosacchariticus. I. Physicochemical characterization. J Biol Chem. 1972 Sep 10;247(17):5596–5601. [PubMed] [Google Scholar]
  12. Markland F. S. Phenolic hydroxyl ionization in two subtilisins. J Biol Chem. 1969 Feb 25;244(4):694–700. [PubMed] [Google Scholar]
  13. Stauffer C. E., Sullivan J. F. The effect of urea and guanidinium chloride on activity of subtilisin Carlsberg. Biochim Biophys Acta. 1971 Dec 28;251(3):407–412. doi: 10.1016/0005-2795(71)90129-2. [DOI] [PubMed] [Google Scholar]
  14. Tachibana A., Murachi T. Phenolic hydroxyl ionization in stem bromelain. Biochemistry. 1966 Aug;5(8):2756–2763. doi: 10.1021/bi00872a036. [DOI] [PubMed] [Google Scholar]
  15. Von Hippel P. H., Wong K. Y. On the conformational stability of globular proteins. The effects of various electrolytes and nonelectrolytes on the thermal ribonuclease transition. J Biol Chem. 1965 Oct;240(10):3909–3923. [PubMed] [Google Scholar]
  16. Wright C. S., Alden R. A., Kraut J. Structure of subtilisin BPN' at 2.5 angström resolution. Nature. 1969 Jan 18;221(5177):235–242. doi: 10.1038/221235a0. [DOI] [PubMed] [Google Scholar]

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