Partially folded intermediates during trypsinogen denaturation
The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was <img SRC="http:/img/fbpe/bjmbr/v32n6/3282sup.gif" ALIGN="BOTTOM" BORDER="0" VSPACE="0&q...
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| Format: | Article |
| Language: | English |
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Associação Brasileira de Divulgação Científica
1999-01-01
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| Series: | Brazilian Journal of Medical and Biological Research |
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| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1999000600002 |
| _version_ | 1818293711737454592 |
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| author | Martins N.F. Santoro M.M. |
| author_facet | Martins N.F. Santoro M.M. |
| author_sort | Martins N.F. |
| collection | DOAJ |
| description | The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was <img SRC="http:/img/fbpe/bjmbr/v32n6/3282sup.gif" ALIGN="BOTTOM" BORDER="0" VSPACE="0" HSPACE="0"> = 6.99 ± 1.40 kcal/mol for guanidine hydrochloride and <img SRC="http:/img/fbpe/bjmbr/v32n6/3282sup.gif" ALIGN="BOTTOM" BORDER="0" VSPACE="0" HSPACE="0"> = 6.37 ± 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 ± 0.4 Å to 26.0 ± 0.3 Å for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 ± 0.3 Å to 25.7 ± 0.6 Å for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics. |
| first_indexed | 2024-12-13T03:20:12Z |
| format | Article |
| id | doaj.art-082633b2226b446bb67ac7d0f38ee710 |
| institution | Directory Open Access Journal |
| issn | 0100-879X 0034-7310 |
| language | English |
| last_indexed | 2024-12-13T03:20:12Z |
| publishDate | 1999-01-01 |
| publisher | Associação Brasileira de Divulgação Científica |
| record_format | Article |
| series | Brazilian Journal of Medical and Biological Research |
| spelling | doaj.art-082633b2226b446bb67ac7d0f38ee7102022-12-22T00:01:23ZengAssociação Brasileira de Divulgação CientíficaBrazilian Journal of Medical and Biological Research0100-879X0034-73101999-01-01326673682Partially folded intermediates during trypsinogen denaturationMartins N.F.Santoro M.M.The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was <img SRC="http:/img/fbpe/bjmbr/v32n6/3282sup.gif" ALIGN="BOTTOM" BORDER="0" VSPACE="0" HSPACE="0"> = 6.99 ± 1.40 kcal/mol for guanidine hydrochloride and <img SRC="http:/img/fbpe/bjmbr/v32n6/3282sup.gif" ALIGN="BOTTOM" BORDER="0" VSPACE="0" HSPACE="0"> = 6.37 ± 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 ± 0.4 Å to 26.0 ± 0.3 Å for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 ± 0.3 Å to 25.7 ± 0.6 Å for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1999000600002trypsinogenprotein denaturationthermodynamic stabilityintermediatesmolten globule |
| spellingShingle | Martins N.F. Santoro M.M. Partially folded intermediates during trypsinogen denaturation Brazilian Journal of Medical and Biological Research trypsinogen protein denaturation thermodynamic stability intermediates molten globule |
| title | Partially folded intermediates during trypsinogen denaturation |
| title_full | Partially folded intermediates during trypsinogen denaturation |
| title_fullStr | Partially folded intermediates during trypsinogen denaturation |
| title_full_unstemmed | Partially folded intermediates during trypsinogen denaturation |
| title_short | Partially folded intermediates during trypsinogen denaturation |
| title_sort | partially folded intermediates during trypsinogen denaturation |
| topic | trypsinogen protein denaturation thermodynamic stability intermediates molten globule |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1999000600002 |
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