Specialized functional domains in hemoglobin: Dimensions in solution of the apohemoglobin dimer labeled with fluorescein iodoacetamide

Massimo Sassaroli, Enrico Bucci, Jerry Liesegang, Clara Fronticelli, Robert F. Steiner

Research output: Contribution to journalArticle

Abstract

The fluorescence characteristics of 8-anilinonaphthalene-1-sulfonic acid (ANS) coupled to apohemoglobin and to apohemoglobin labeled with fluorescein iodoacetamide (FIA) at β-93 have been compared. The quenching of emission of ANS produced by FIA was measured both with steady-state and with time-resolved techniques. In this system the emission of ANS in the β-heme pockets was totally quenched by FIA at β-93. Steady-state measurements indicated a 57% efficiency of energy transfer between ANS in the α-heme pockets and FIA at β-93. Time resolution showed that the initial (unquenched) lifetime of ANS was 18.2 ns. In the presence of FIA two new components were generated with lifetimes of 2.0 and 6.6 ns. Assuming a random orientation of the probes, the distances inferred from these measurements were near 4.6 and 3.6 nm for the time-resolved and near 48 Å for the steady-state measurements. In the tridimensional model of hemoglobin the distance between the iron atom of the α1 chains and the SH group of the β1, chains at position 93 is 3.6 nm in oxyhemoglobin and 4.1 nm in deoxyhemoglobin. To these distances 0.5-1.0 nm may be added to allow for the dimensions of the probes. Thus it appears that removal of the heme fails to produce any important enlargement of the molecule. On the contrary, the data suggest a slight shrinking of apohemoglobin, which may be consistent with a collapse of the heme pocket when heme is removed. The rest of the molecule does not seem to be greatly affected. These observations suggest that the modification of the secondary structure of hemoglobin upon removal of heme is an event that specifically regulates the conformation of the α1β2 interface, which is broken in apohemoglobin. This gives further support to the hypothesis that in hemoglobin domains of secondary structures specifically store and transmit to functional interfaces information with regard to the state of the heme.

Original languageEnglish (US)
Pages (from-to)2487-2491
Number of pages5
JournalBiochemistry®
Volume23
Issue number11
StatePublished - 1984
Externally publishedYes

Fingerprint

Heme
Dimers
Hemoglobins
Oxyhemoglobins
Molecules
Energy Transfer
apohemoglobin
5-iodoacetamidofluorescein
Energy transfer
Conformations
Quenching
Iron
Fluorescence
1-anilino-8-naphthalenesulfonate
Atoms

ASJC Scopus subject areas

  • Biochemistry

Cite this

Specialized functional domains in hemoglobin : Dimensions in solution of the apohemoglobin dimer labeled with fluorescein iodoacetamide. / Sassaroli, Massimo; Bucci, Enrico; Liesegang, Jerry; Fronticelli, Clara; Steiner, Robert F.

In: Biochemistry®, Vol. 23, No. 11, 1984, p. 2487-2491.

Research output: Contribution to journalArticle

Sassaroli, Massimo ; Bucci, Enrico ; Liesegang, Jerry ; Fronticelli, Clara ; Steiner, Robert F. / Specialized functional domains in hemoglobin : Dimensions in solution of the apohemoglobin dimer labeled with fluorescein iodoacetamide. In: Biochemistry®. 1984 ; Vol. 23, No. 11. pp. 2487-2491.
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abstract = "The fluorescence characteristics of 8-anilinonaphthalene-1-sulfonic acid (ANS) coupled to apohemoglobin and to apohemoglobin labeled with fluorescein iodoacetamide (FIA) at β-93 have been compared. The quenching of emission of ANS produced by FIA was measured both with steady-state and with time-resolved techniques. In this system the emission of ANS in the β-heme pockets was totally quenched by FIA at β-93. Steady-state measurements indicated a 57{\%} efficiency of energy transfer between ANS in the α-heme pockets and FIA at β-93. Time resolution showed that the initial (unquenched) lifetime of ANS was 18.2 ns. In the presence of FIA two new components were generated with lifetimes of 2.0 and 6.6 ns. Assuming a random orientation of the probes, the distances inferred from these measurements were near 4.6 and 3.6 nm for the time-resolved and near 48 {\AA} for the steady-state measurements. In the tridimensional model of hemoglobin the distance between the iron atom of the α1 chains and the SH group of the β1, chains at position 93 is 3.6 nm in oxyhemoglobin and 4.1 nm in deoxyhemoglobin. To these distances 0.5-1.0 nm may be added to allow for the dimensions of the probes. Thus it appears that removal of the heme fails to produce any important enlargement of the molecule. On the contrary, the data suggest a slight shrinking of apohemoglobin, which may be consistent with a collapse of the heme pocket when heme is removed. The rest of the molecule does not seem to be greatly affected. These observations suggest that the modification of the secondary structure of hemoglobin upon removal of heme is an event that specifically regulates the conformation of the α1β2 interface, which is broken in apohemoglobin. This gives further support to the hypothesis that in hemoglobin domains of secondary structures specifically store and transmit to functional interfaces information with regard to the state of the heme.",
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AB - The fluorescence characteristics of 8-anilinonaphthalene-1-sulfonic acid (ANS) coupled to apohemoglobin and to apohemoglobin labeled with fluorescein iodoacetamide (FIA) at β-93 have been compared. The quenching of emission of ANS produced by FIA was measured both with steady-state and with time-resolved techniques. In this system the emission of ANS in the β-heme pockets was totally quenched by FIA at β-93. Steady-state measurements indicated a 57% efficiency of energy transfer between ANS in the α-heme pockets and FIA at β-93. Time resolution showed that the initial (unquenched) lifetime of ANS was 18.2 ns. In the presence of FIA two new components were generated with lifetimes of 2.0 and 6.6 ns. Assuming a random orientation of the probes, the distances inferred from these measurements were near 4.6 and 3.6 nm for the time-resolved and near 48 Å for the steady-state measurements. In the tridimensional model of hemoglobin the distance between the iron atom of the α1 chains and the SH group of the β1, chains at position 93 is 3.6 nm in oxyhemoglobin and 4.1 nm in deoxyhemoglobin. To these distances 0.5-1.0 nm may be added to allow for the dimensions of the probes. Thus it appears that removal of the heme fails to produce any important enlargement of the molecule. On the contrary, the data suggest a slight shrinking of apohemoglobin, which may be consistent with a collapse of the heme pocket when heme is removed. The rest of the molecule does not seem to be greatly affected. These observations suggest that the modification of the secondary structure of hemoglobin upon removal of heme is an event that specifically regulates the conformation of the α1β2 interface, which is broken in apohemoglobin. This gives further support to the hypothesis that in hemoglobin domains of secondary structures specifically store and transmit to functional interfaces information with regard to the state of the heme.

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