DOI: 10.1111/j.1432-1033.1974.tb03295.x. A Spectroscopic Study of the Haemin

A Spectroscopic Study of the Haemin–Human‐Serum‐Albumin System

10.1111/j.1432-1033.1974.tb03295.x

Crossref journal-article

Wiley

European Journal of Biochemistry (311)

Abstract

The interaction of haemin with human serum albumin has been reexamined. The absorption spectrum of the bound haem is identical with that of uncomplexed monomeric haemin in solution, and it is suggested, on the basis of an interaction of albumin with iron‐free protoporphyrin IX, that the iron is not implicated in the interaction with the protein. A ferric cyanide derivative, and a ferrous haem derivative of methaemalbumin can be recognised, but not azide or fluoride derivatives. The bound haemin gives rise to extrinsic Cotton effects, which are different in detail in the derivatives, and in the complex with protoporphyrin IX Spectrophotometric titrations show that there is one strong binding site for haemin and several weaker sites. The latter are associated with optical activity opposite in sign to that of the strong complex. The binding profiles are little affected by pH over a wide range, by ionic strength or by the presence of 40% (v/v) dimethylsulphoxide/water, in which the free haemin is monomeric. The binding of haemin to albumin has been followed by spectrophotometry, circular dichroism and fluorescence. The binding of haemin quenches the protein fluorescence, which progressively changes in character from tryptophan to tyrosine type. The haem at the primary binding site thus strongly quenches the tryptophan specifically. From fluorescence titrations at a range of protein concentrations, binding isotherms were constructed, and gave an association constant for the strong site of 50 μM−1. From binding isotherms based on absorption measurements we can infer the existence of at least four sites with much lower binding constants. A long‐chain fatty acid anion was found to compete with haemin only for the weaker binding sites, so that binding beyond one mole per mole of protein can be essentially eliminated. The open‐chain tetrapyrrole, bilirubin, was found, in agreement with earlier work, not to compete with haemin, as regards the strongest binding sites of either ligand. Between the weaker sites, however, evidence of competition was obtained.

Bibliography

Beaven, G. H., Chen, S., Dâalbis, A., & Gratzer, W. B. (1974). A Spectroscopic Study of the HaeminâHumanâSerumâAlbumin System. European Journal of Biochemistry, 41(3), 539â546. Portico.

Authors 4

Gilbert H. Beaven (first)
Shi‐Hua Chen (additional)
Anne D'albis (additional)
Walter B. Gratzer (additional)

References 29 Referenced 257

10.1016/S0021-9258(19)51193-0
10.1042/bj0660552
{'key': 'e_1_2_1_4_2', 'first-page': '95', 'volume': '10', 'author': 'Fairley N. H.', 'year': '1941', 'journal-title': 'Q. J. Med.'} / Q. J. Med. by Fairley N. H. (1941)
10.1136/bmj.2.5054.1137
10.1016/S0021-9258(17)41115-X
10.1016/0006-291X(62)90048-7
10.1016/S0021-9258(19)81445-X
{'key': 'e_1_2_1_9_2', 'first-page': '316', 'volume-title': 'Data for Biochemical Research', 'author': 'Dawson R. M. C.', 'year': '1969'} / Data for Biochemical Research by Dawson R. M. C. (1969)
{'key': 'e_1_2_1_10_2', 'first-page': '126', 'volume-title': 'Porphyrins and Metalloporphyrins', 'author': 'Falk J. E.', 'year': '1964'} / Porphyrins and Metalloporphyrins by Falk J. E. (1964)
10.1016/S0021-9258(18)63402-7
10.1111/j.1432-1033.1973.tb02709.x
10.1021/bi00768a025
10.1111/j.1749-6632.1949.tb27297.x
10.1042/bj1150279
10.1016/0005-2795(72)90108-0
10.1038/192630a0
10.1016/B978-1-4832-2810-5.50013-X
10.1039/df9592700134
10.1042/bj0760381
{'key': 'e_1_2_1_21_2', 'first-page': '547', 'volume': '79', 'author': 'Blauer G.', 'year': '1964', 'journal-title': 'Biochim. Biophys. Acta'} / Biochim. Biophys. Acta by Blauer G. (1964)
{'key': 'e_1_2_1_22_2', 'first-page': '330', 'volume-title': 'Poly‐α‐Amino Acids', 'author': 'Beychock S.', 'year': '1967'} / Poly‐α‐Amino Acids by Beychock S. (1967)
10.1002/9780470143506.ch2
10.1021/ja01041a062
10.1016/0006-3002(57)90470-5
10.1021/bi00815a003
10.1021/ja01548a024
10.1021/bi00798a001
10.1016/0003-9861(70)90023-8
10.1016/0006-291X(71)90535-3

Dates

Type	When
Created	20 years, 6 months ago (March 4, 2005, 7:46 a.m.)
Deposited	1 year, 11 months ago (Oct. 1, 2023, 2:17 p.m.)
Indexed	3 days, 2 hours ago (Sept. 4, 2025, 10:24 a.m.)
Issued	51 years, 7 months ago (Feb. 1, 1974)
Published	51 years, 7 months ago (Feb. 1, 1974)
Published Online	20 years, 6 months ago (March 3, 2005)
Published Print	51 years, 7 months ago (Feb. 1, 1974)

Funders 0

None

BibTeX

@article{Beaven_1974, title={A Spectroscopic Study of the Haemin–Human‐Serum‐Albumin System}, volume={41}, ISSN={1432-1033}, url={http://dx.doi.org/10.1111/j.1432-1033.1974.tb03295.x}, DOI={10.1111/j.1432-1033.1974.tb03295.x}, number={3}, journal={European Journal of Biochemistry}, publisher={Wiley}, author={Beaven, Gilbert H. and Chen, Shi‐Hua and D’albis, Anne and Gratzer, Walter B.}, year={1974}, month=feb, pages={539–546} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        9,
        4
      ]
    ],
    "date-time": "2025-09-04T14:24:20Z",
    "timestamp": 1756995860607
  },
  "reference-count": 29,
  "publisher": "Wiley",
  "issue": "3",
  "license": [
    {
      "start": {
        "date-parts": [
          [
            2005,
            3,
            3
          ]
        ],
        "date-time": "2005-03-03T00:00:00Z",
        "timestamp": 1109808000000
      },
      "content-version": "vor",
      "delay-in-days": 11353,
      "URL": "http://onlinelibrary.wiley.com/termsAndConditions#vor"
    }
  ],
  "content-domain": {
    "domain": [],
    "crossmark-restriction": false
  },
  "published-print": {
    "date-parts": [
      [
        1974,
        2
      ]
    ]
  },
  "abstract": "<jats:p>The interaction of haemin with human serum albumin has been reexamined. The absorption spectrum of the bound haem is identical with that of uncomplexed monomeric haemin in solution, and it is suggested, on the basis of an interaction of albumin with iron\u2010free protoporphyrin IX, that the iron is not implicated in the interaction with the protein. A ferric cyanide derivative, and a ferrous haem derivative of methaemalbumin can be recognised, but not azide or fluoride derivatives. The bound haemin gives rise to extrinsic Cotton effects, which are different in detail in the derivatives, and in the complex with protoporphyrin IX Spectrophotometric titrations show that there is one strong binding site for haemin and several weaker sites. The latter are associated with optical activity opposite in sign to that of the strong complex. The binding profiles are little affected by pH over a wide range, by ionic strength or by the presence of 40% (v/v) dimethylsulphoxide/water, in which the free haemin is monomeric. The binding of haemin to albumin has been followed by spectrophotometry, circular dichroism and fluorescence. The binding of haemin quenches the protein fluorescence, which progressively changes in character from tryptophan to tyrosine type. The haem at the primary binding site thus strongly quenches the tryptophan specifically. From fluorescence titrations at a range of protein concentrations, binding isotherms were constructed, and gave an association constant for the strong site of 50 \u03bcM<jats:sup>\u22121</jats:sup>. From binding isotherms based on absorption measurements we can infer the existence of at least four sites with much lower binding constants. A long\u2010chain fatty acid anion was found to compete with haemin only for the weaker binding sites, so that binding beyond one mole per mole of protein can be essentially eliminated. The open\u2010chain tetrapyrrole, bilirubin, was found, in agreement with earlier work, not to compete with haemin, as regards the strongest binding sites of either ligand. Between the weaker sites, however, evidence of competition was obtained.</jats:p>",
  "DOI": "10.1111/j.1432-1033.1974.tb03295.x",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2005,
        3,
        4
      ]
    ],
    "date-time": "2005-03-04T12:46:09Z",
    "timestamp": 1109940369000
  },
  "page": "539-546",
  "source": "Crossref",
  "is-referenced-by-count": 257,
  "title": "A Spectroscopic Study of the Haemin\u2013Human\u2010Serum\u2010Albumin System",
  "prefix": "10.1111",
  "volume": "41",
  "author": [
    {
      "given": "Gilbert H.",
      "family": "Beaven",
      "sequence": "first",
      "affiliation": []
    },
    {
      "given": "Shi\u2010Hua",
      "family": "Chen",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Anne",
      "family": "D'albis",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Walter B.",
      "family": "Gratzer",
      "sequence": "additional",
      "affiliation": []
    }
  ],
  "member": "311",
  "published-online": {
    "date-parts": [
      [
        2005,
        3,
        3
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_2_1_2_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(19)51193-0"
    },
    {
      "key": "e_1_2_1_3_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1042/bj0660552"
    },
    {
      "key": "e_1_2_1_4_2",
      "first-page": "95",
      "volume": "10",
      "author": "Fairley N. H.",
      "year": "1941",
      "journal-title": "Q. J. Med."
    },
    {
      "key": "e_1_2_1_5_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1136/bmj.2.5054.1137"
    },
    {
      "key": "e_1_2_1_6_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(17)41115-X"
    },
    {
      "key": "e_1_2_1_7_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0006-291X(62)90048-7"
    },
    {
      "key": "e_1_2_1_8_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(19)81445-X"
    },
    {
      "key": "e_1_2_1_9_2",
      "first-page": "316",
      "volume-title": "Data for Biochemical Research",
      "author": "Dawson R. M. C.",
      "year": "1969"
    },
    {
      "key": "e_1_2_1_10_2",
      "first-page": "126",
      "volume-title": "Porphyrins and Metalloporphyrins",
      "author": "Falk J. E.",
      "year": "1964"
    },
    {
      "key": "e_1_2_1_11_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(18)63402-7"
    },
    {
      "key": "e_1_2_1_12_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1432-1033.1973.tb02709.x"
    },
    {
      "key": "e_1_2_1_13_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00768a025"
    },
    {
      "key": "e_1_2_1_14_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1749-6632.1949.tb27297.x"
    },
    {
      "key": "e_1_2_1_15_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1042/bj1150279"
    },
    {
      "key": "e_1_2_1_16_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0005-2795(72)90108-0"
    },
    {
      "key": "e_1_2_1_17_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/192630a0"
    },
    {
      "key": "e_1_2_1_18_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/B978-1-4832-2810-5.50013-X"
    },
    {
      "key": "e_1_2_1_19_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1039/df9592700134"
    },
    {
      "key": "e_1_2_1_20_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1042/bj0760381"
    },
    {
      "key": "e_1_2_1_21_2",
      "first-page": "547",
      "volume": "79",
      "author": "Blauer G.",
      "year": "1964",
      "journal-title": "Biochim. Biophys. Acta"
    },
    {
      "key": "e_1_2_1_22_2",
      "first-page": "330",
      "volume-title": "Poly\u2010\u03b1\u2010Amino Acids",
      "author": "Beychock S.",
      "year": "1967"
    },
    {
      "key": "e_1_2_1_23_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/9780470143506.ch2"
    },
    {
      "key": "e_1_2_1_24_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/ja01041a062"
    },
    {
      "key": "e_1_2_1_25_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0006-3002(57)90470-5"
    },
    {
      "key": "e_1_2_1_26_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00815a003"
    },
    {
      "key": "e_1_2_1_27_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/ja01548a024"
    },
    {
      "key": "e_1_2_1_28_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00798a001"
    },
    {
      "key": "e_1_2_1_29_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0003-9861(70)90023-8"
    },
    {
      "key": "e_1_2_1_30_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0006-291X(71)90535-3"
    }
  ],
  "container-title": "European Journal of Biochemistry",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fj.1432-1033.1974.tb03295.x",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "text-mining"
    },
    {
      "URL": "https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1974.tb03295.x",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2023,
        10,
        1
      ]
    ],
    "date-time": "2023-10-01T18:17:03Z",
    "timestamp": 1696184223000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1974.tb03295.x"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        1974,
        2
      ]
    ]
  },
  "references-count": 29,
  "journal-issue": {
    "issue": "3",
    "published-print": {
      "date-parts": [
        [
          1974,
          2
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1111/j.1432-1033.1974.tb03295.x"
  ],
  "URL": "http://dx.doi.org/10.1111/j.1432-1033.1974.tb03295.x",
  "relation": {},
  "ISSN": [
    "0014-2956",
    "1432-1033"
  ],
  "subject": [],
  "container-title-short": "European Journal of Biochemistry",
  "published": {
    "date-parts": [
      [
        1974,
        2
      ]
    ]
  }
}