DOI: 10.1111/j.1432-1033.1996.0862p.x. Thermal Unfolding and Proteolytic Susceptibility of Ribonuclease A

Thermal Unfolding and Proteolytic Susceptibility of Ribonuclease A

10.1111/j.1432-1033.1996.0862p.x

Crossref journal-article

Wiley

European Journal of Biochemistry (311)

Abstract

With the aim to localize the structural region that becomes first accessible to proteolytic attack during thermal unfolding, the proteolysis of ribonuclease A was studied in the temperature range of 20–65°C. Subtilisin, proteinase K, and elastase proved to be not appropriate as indicators of thermal unfolding, because even the native protein molecule was cleaved by these proteases. In contrast, chymotrypsin, trypsin, and thermolysin attacked ribonuclease A only after its thermal treatment. For thermolysin and trypsin, the first primary cleavage sites of ribonuclease A could be identified by blotting of the electropho‐retic bands, partial N‐terminal sequencing of the fragments and assignment according to their molecular masses. The results were confirmed by the separation of the proteolytic fragments by HPLC and subsequent matrix‐assisted laser desorption ionization mass spectrometry. The first cleavage sites were determined to be Lys31‐Ser32 and Arg33‐Asn34 for trypsin and Asn34‐Leu35 and Thr45‐Phe46 for thermolysin. Hence the structural region from Lys31 to Leu35, together with the adjacent β‐structure containing Thr45‐Phe46, is suggested to represent a labile region of the ribonuclease A molecule, which becomes exposed at thermal denaturation.

Bibliography

Arnold, U., RÃ¼cknagel, K. P., Schierhorn, A., & UlbrichâHofmann, R. (1996). Thermal Unfolding and Proteolytic Susceptibility of Ribonuclease A. European Journal of Biochemistry, 237(3), 862â869. Portico.

Authors 4

Ulrich Arnold (first)
Karl Peter Rücknagel (additional)
Angelika Schierhorn (additional)
Renate Ulbrich‐Hofmann (additional)

References 45 Referenced 62

10.1021/bi00407a033
{'key': 'e_1_2_3_3_1', 'first-page': '43', 'volume-title': 'Protein sequencing. A practical approach', 'author': 'Aitken A.', 'year': '1989'} / Protein sequencing. A practical approach by Aitken A. (1989)
10.1016/S0021-9258(18)99563-3 / J. Biol. Chem. / Evidence for residual structure in acid‐ and heat‐denatured proteins by Aune K. C. (1967)
10.1002/elps.1150080203
10.1016/S0022-5193(75)80012-9
10.1021/bi00654a015
10.1016/0022-2836(79)90504-7
{'key': 'e_1_2_3_9_1', 'volume-title': 'Thermostability of enzymes', 'author': 'Gupta N. N.', 'year': '1993'} / Thermostability of enzymes by Gupta N. N. (1993)
10.1021/bi00175a022
10.1016/0005-2795(79)90495-1
10.1002/pro.5560030505
10.1016/0022-2836(86)90250-0
10.1111/j.1432-1033.1993.tb18370.x
10.1073/pnas.92.7.2657
10.1038/375513a0
10.1016/S0021-9258(18)97265-0 / J. Biol. Chem. / Ribonuclease E, an intermediate in the degradation of ribonuclease by porcine elastase by Klee W. A. (1965)
10.1021/bi00864a016
10.1016/0022-2836(82)90238-8
10.1111/j.1432-1033.1986.tb09864.x
10.1126/science.8235609
10.1021/ar00158a004
10.1021/bi00903a006
10.1073/pnas.92.16.7292
{'key': 'e_1_2_3_25_1', 'first-page': '163', 'volume-title': 'Proteolytic enzymes. A practical approach', 'author': 'Price N. C.', 'year': '1990'} / Proteolytic enzymes. A practical approach by Price N. C. (1990)
10.1016/0022-2836(89)90318-5
10.1016/S1874-6047(08)60384-4
10.1111/j.1749-6632.1973.tb15257.x
10.1021/bi00105a014
10.1021/bi00903a005
10.1016/0003-2697(87)90587-2
10.1002/pro.5560010710
10.1021/bi00172a010
10.1016/S0021-9258(19)83984-4
10.1021/bi00200a027
10.1021/bi00006a008
10.1021/bi00815a015
10.1002/bit.260280407
10.1016/B978-0-444-89372-7.50065-8 / Stability and stabilization of enzymes by Ulbrich‐Hofman R. (1993)
{'key': 'e_1_2_3_40_1', 'first-page': '75', 'article-title': 'Structural flexibility in extremely stable carrier‐bound chymotrypsin', 'volume': '22', 'author': 'Ulbrich‐Hofmann R.', 'year': '1995', 'journal-title': 'Biotechnol. Appl. Biochem.'} / Biotechnol. Appl. Biochem. / Structural flexibility in extremely stable carrier‐bound chymotrypsin by Ulbrich‐Hofmann R. (1995)
{'key': 'e_1_2_3_41_1', 'volume-title': 'Stability and stabilization of enzymes', 'author': 'Tweel W. J. J.', 'year': '1993'} / Stability and stabilization of enzymes by Tweel W. J. J. (1993)
10.1042/bj1170299
10.1016/S0021-9258(19)68195-0 / J. Biol. Chem. / The refined crystal structure of ribonuclease A at 2.0 A by Wlodawer A. (1982)
10.1006/jmbi.1995.0408
10.1042/bj3050379
10.1021/bi00027a012

Dates

Type	When
Created	21 years ago (Aug. 31, 2004, 10:14 a.m.)
Deposited	1 year, 10 months ago (Oct. 31, 2023, 7:13 a.m.)
Indexed	1 month, 3 weeks ago (July 4, 2025, 7:48 a.m.)
Issued	29 years, 4 months ago (May 1, 1996)
Published	29 years, 4 months ago (May 1, 1996)
Published Online	21 years ago (Aug. 31, 2004)
Published Print	29 years, 4 months ago (May 1, 1996)

Funders 0

None

BibTeX

@article{Arnold_1996, title={Thermal Unfolding and Proteolytic Susceptibility of Ribonuclease A}, volume={237}, ISSN={1432-1033}, url={http://dx.doi.org/10.1111/j.1432-1033.1996.0862p.x}, DOI={10.1111/j.1432-1033.1996.0862p.x}, number={3}, journal={European Journal of Biochemistry}, publisher={Wiley}, author={Arnold, Ulrich and Rücknagel, Karl Peter and Schierhorn, Angelika and Ulbrich‐Hofmann, Renate}, year={1996}, month=may, pages={862–869} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        7,
        4
      ]
    ],
    "date-time": "2025-07-04T11:48:01Z",
    "timestamp": 1751629681327
  },
  "reference-count": 45,
  "publisher": "Wiley",
  "issue": "3",
  "license": [
    {
      "start": {
        "date-parts": [
          [
            2004,
            8,
            31
          ]
        ],
        "date-time": "2004-08-31T00:00:00Z",
        "timestamp": 1093910400000
      },
      "content-version": "vor",
      "delay-in-days": 3044,
      "URL": "http://onlinelibrary.wiley.com/termsAndConditions#vor"
    }
  ],
  "content-domain": {
    "domain": [],
    "crossmark-restriction": false
  },
  "published-print": {
    "date-parts": [
      [
        1996,
        5
      ]
    ]
  },
  "abstract": "<jats:p>With the aim to localize the structural region that becomes first accessible to proteolytic attack during thermal unfolding, the proteolysis of ribonuclease A was studied in the temperature range of 20\u201365\u00b0C. Subtilisin, proteinase K, and elastase proved to be not appropriate as indicators of thermal unfolding, because even the native protein molecule was cleaved by these proteases. In contrast, chymotrypsin, trypsin, and thermolysin attacked ribonuclease A only after its thermal treatment. For thermolysin and trypsin, the first primary cleavage sites of ribonuclease A could be identified by blotting of the electropho\u2010retic bands, partial N\u2010terminal sequencing of the fragments and assignment according to their molecular masses. The results were confirmed by the separation of the proteolytic fragments by HPLC and subsequent matrix\u2010assisted laser desorption ionization mass spectrometry. The first cleavage sites were determined to be Lys31\u2010Ser32 and Arg33\u2010Asn34 for trypsin and Asn34\u2010Leu35 and Thr45\u2010Phe46 for thermolysin. Hence the structural region from Lys31 to Leu35, together with the adjacent \u03b2\u2010structure containing Thr45\u2010Phe46, is suggested to represent a labile region of the ribonuclease A molecule, which becomes exposed at thermal denaturation.</jats:p>",
  "DOI": "10.1111/j.1432-1033.1996.0862p.x",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2004,
        8,
        31
      ]
    ],
    "date-time": "2004-08-31T14:14:04Z",
    "timestamp": 1093961644000
  },
  "page": "862-869",
  "source": "Crossref",
  "is-referenced-by-count": 62,
  "title": "Thermal Unfolding and Proteolytic Susceptibility of Ribonuclease A",
  "prefix": "10.1111",
  "volume": "237",
  "author": [
    {
      "given": "Ulrich",
      "family": "Arnold",
      "sequence": "first",
      "affiliation": []
    },
    {
      "given": "Karl Peter",
      "family": "R\u00fccknagel",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Angelika",
      "family": "Schierhorn",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Renate",
      "family": "Ulbrich\u2010Hofmann",
      "sequence": "additional",
      "affiliation": []
    }
  ],
  "member": "311",
  "published-online": {
    "date-parts": [
      [
        2004,
        8,
        31
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_2_3_2_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00407a033"
    },
    {
      "key": "e_1_2_3_3_1",
      "first-page": "43",
      "volume-title": "Protein sequencing. A practical approach",
      "author": "Aitken A.",
      "year": "1989"
    },
    {
      "key": "e_1_2_3_4_1",
      "doi-asserted-by": "crossref",
      "first-page": "4486",
      "DOI": "10.1016/S0021-9258(18)99563-3",
      "article-title": "Evidence for residual structure in acid\u2010 and heat\u2010denatured proteins",
      "volume": "242",
      "author": "Aune K. C.",
      "year": "1967",
      "journal-title": "J. Biol. Chem."
    },
    {
      "key": "e_1_2_3_5_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/elps.1150080203"
    },
    {
      "key": "e_1_2_3_6_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0022-5193(75)80012-9"
    },
    {
      "key": "e_1_2_3_7_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00654a015"
    },
    {
      "key": "e_1_2_3_8_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0022-2836(79)90504-7"
    },
    {
      "key": "e_1_2_3_9_1",
      "volume-title": "Thermostability of enzymes",
      "author": "Gupta N. N.",
      "year": "1993"
    },
    {
      "key": "e_1_2_3_10_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00175a022"
    },
    {
      "key": "e_1_2_3_11_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0005-2795(79)90495-1"
    },
    {
      "key": "e_1_2_3_12_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/pro.5560030505"
    },
    {
      "key": "e_1_2_3_13_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0022-2836(86)90250-0"
    },
    {
      "key": "e_1_2_3_14_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1432-1033.1993.tb18370.x"
    },
    {
      "key": "e_1_2_3_15_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.92.7.2657"
    },
    {
      "key": "e_1_2_3_16_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/375513a0"
    },
    {
      "key": "e_1_2_3_17_1",
      "doi-asserted-by": "crossref",
      "first-page": "2900",
      "DOI": "10.1016/S0021-9258(18)97265-0",
      "article-title": "Ribonuclease E, an intermediate in the degradation of ribonuclease by porcine elastase",
      "volume": "240",
      "author": "Klee W. A.",
      "year": "1965",
      "journal-title": "J. Biol. Chem."
    },
    {
      "key": "e_1_2_3_18_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00864a016"
    },
    {
      "key": "e_1_2_3_19_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0022-2836(82)90238-8"
    },
    {
      "key": "e_1_2_3_20_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1432-1033.1986.tb09864.x"
    },
    {
      "key": "e_1_2_3_21_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.8235609"
    },
    {
      "key": "e_1_2_3_22_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/ar00158a004"
    },
    {
      "key": "e_1_2_3_23_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00903a006"
    },
    {
      "key": "e_1_2_3_24_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.92.16.7292"
    },
    {
      "key": "e_1_2_3_25_1",
      "first-page": "163",
      "volume-title": "Proteolytic enzymes. A practical approach",
      "author": "Price N. C.",
      "year": "1990"
    },
    {
      "key": "e_1_2_3_26_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0022-2836(89)90318-5"
    },
    {
      "key": "e_1_2_3_27_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S1874-6047(08)60384-4"
    },
    {
      "key": "e_1_2_3_28_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1749-6632.1973.tb15257.x"
    },
    {
      "key": "e_1_2_3_29_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00105a014"
    },
    {
      "key": "e_1_2_3_30_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00903a005"
    },
    {
      "key": "e_1_2_3_31_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0003-2697(87)90587-2"
    },
    {
      "key": "e_1_2_3_32_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/pro.5560010710"
    },
    {
      "key": "e_1_2_3_33_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00172a010"
    },
    {
      "key": "e_1_2_3_34_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(19)83984-4"
    },
    {
      "key": "e_1_2_3_35_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00200a027"
    },
    {
      "key": "e_1_2_3_36_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00006a008"
    },
    {
      "key": "e_1_2_3_37_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00815a015"
    },
    {
      "key": "e_1_2_3_38_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/bit.260280407"
    },
    {
      "key": "e_1_2_3_39_1",
      "doi-asserted-by": "crossref",
      "first-page": "497",
      "DOI": "10.1016/B978-0-444-89372-7.50065-8",
      "volume-title": "Stability and stabilization of enzymes",
      "author": "Ulbrich\u2010Hofman R.",
      "year": "1993"
    },
    {
      "key": "e_1_2_3_40_1",
      "first-page": "75",
      "article-title": "Structural flexibility in extremely stable carrier\u2010bound chymotrypsin",
      "volume": "22",
      "author": "Ulbrich\u2010Hofmann R.",
      "year": "1995",
      "journal-title": "Biotechnol. Appl. Biochem."
    },
    {
      "key": "e_1_2_3_41_1",
      "volume-title": "Stability and stabilization of enzymes",
      "author": "Tweel W. J. J.",
      "year": "1993"
    },
    {
      "key": "e_1_2_3_42_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1042/bj1170299"
    },
    {
      "key": "e_1_2_3_43_1",
      "doi-asserted-by": "crossref",
      "first-page": "1325",
      "DOI": "10.1016/S0021-9258(19)68195-0",
      "article-title": "The refined crystal structure of ribonuclease A at 2.0 A",
      "volume": "257",
      "author": "Wlodawer A.",
      "year": "1982",
      "journal-title": "J. Biol. Chem."
    },
    {
      "key": "e_1_2_3_44_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1006/jmbi.1995.0408"
    },
    {
      "key": "e_1_2_3_45_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1042/bj3050379"
    },
    {
      "key": "e_1_2_3_46_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00027a012"
    }
  ],
  "container-title": "European Journal of Biochemistry",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fj.1432-1033.1996.0862p.x",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "text-mining"
    },
    {
      "URL": "https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1996.0862p.x",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2023,
        10,
        31
      ]
    ],
    "date-time": "2023-10-31T11:13:13Z",
    "timestamp": 1698750793000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1996.0862p.x"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        1996,
        5
      ]
    ]
  },
  "references-count": 45,
  "journal-issue": {
    "issue": "3",
    "published-print": {
      "date-parts": [
        [
          1996,
          5
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1111/j.1432-1033.1996.0862p.x"
  ],
  "URL": "http://dx.doi.org/10.1111/j.1432-1033.1996.0862p.x",
  "relation": {},
  "ISSN": [
    "0014-2956",
    "1432-1033"
  ],
  "subject": [],
  "container-title-short": "European Journal of Biochemistry",
  "published": {
    "date-parts": [
      [
        1996,
        5
      ]
    ]
  }
}