Het doel van het onderzoek is de bestudering van de verandering van eiwitmolekulen onder invloed van verschillende agentia. Hierbij hebben we ons beperkt tot twee eiwitten, ferritine en hemocyanine. Dit werk vormt een deel van het onderzoek van de "Werkgemeenschap Eiwitten"van de Stichting "Scheikundig Onderzoek Nederland" en werd mede mogelijk gemaakt door geldelijke steun van de Nederlandse Organisatie voor Zuiver Wetenschappelijk Onderzoek (ZWO) ... Zie: Summar

Bruggen, Ernst Frederik Jacobus van

English

University of Groningen Research Database

  
 University of Groningen
Elektronenmicroscopie van ferritine en hemocyanine
Bruggen, Ernst Frederik Jacobus van
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
1962
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Bruggen, E. F. J. V. (1962). Elektronenmicroscopie van ferritine en hemocyanine s.n.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 11-02-2018
Summary 
ELECTRON MICROSCOPY OF FERRITIN AND 
HEMOCYANIN 
1. INTRODUCTION 
The concepts primary, secondary, tertiary and quarternary struc- 
ture of proteins are described. Electron microscopy chiefly deals with 
the quarternary structure: the way protein molecules are built from 
subunits. 
By electron microscopy we have studied the structural changes of 
ferritin and hemocyanin molecules under the influence of different 
chemical agents. 
2. TECHNIQUES 
The preparation of carbon films and the way protein molecules 
are sprayed onto the carbon film are described. The various staining 
methods for proteins can be divided in shadow-techniques, positive- 
and negative-staining methods. Several negative-staining agents are 
discussed e.g. potassium phosphotungstate (PTA), sodium tungstate, 
uranyl acetate and a complex of uranyl acetate with ethylene 
diamine tetraacetate ("uranyl. EDTA-complex"). 
Use was made of the Siemens Elmiskop-I and the Philips EM-100 
and EM-200 electron microscopes. The beam voltages were between 
40 and 80 kV, the "spot" size was 15-25 ,U, the emission current 
about 20 pA and the diameter of the objective aperture 25-50 p. 
The astigmatism of the objective lens was reduced to less then 0.2 ,L[. 
Photographs were taken on Ilford 5Bll  35 mm film at  a magni- 
fication of 18,000-35,000 X and on Ilford Special Lantern Plates 
(gradation: contrasty) at a magnification of 40,000-80,000 X. The 
films were developed in a ten times diluted Glycinool developer 
(make: Amaloco), the plates in Kodak D61A developer for 15 and 
3 minutes respectively. The developing temperature was 18-20' C. 
3. FERRITIN 
Ferritin is a remarkable protein, because of its high iron content 
(about 20 Olo of its dry weight). The iron is concentrated in a micelle 
of composition (FeO.OH),(FeO.OPO,H,). When the iron is removed 
from ferritin by the action of reducing agents apoferritin is formed. 
b I t  is possible to introduce some iron again into apoferritin, by which 
a "synthetic ferritin" is obtained. 
A short survey is given of the literature on ferritin. I t  was shown 
previously by electron microscopy that a ferritin molecule consists of 
the iron containing micelle surrounded by an approximately spherical 
shell of apoferritin. 
We studied the structure of the ferritin molecule in some detail, 
the intermediate stages of the molecules during the removal of the 
iron, the apoferritin molecule and the molecule of "synthetib fer- 
ritin". Use was made of the replica and shadow-technique of HALL 
h and several negative-staining methods. 
A model is given for the iron-containing micelle; it  consists of 
6 subunits at the corners of a trilateral prism (pointgroup symmetry 
- 
6). The structure of apoferritin could not be elucidated, although an 
indication of the arrangement of the subunits has been obtained. 
The centre of the apoferritin molecule is hollow and filled with the 
surrounding solution. 
In Table I11 the FeIN-weight ratios of ferritin, apoferritin and 
the intermediate stages are given. The proportions of the number 
of full, partly full and empty molecules as observed on electron 
micrographs are listed for each Fe/N-ratio. The results are also 
shown in Figure I. I t  is concluded that the removal of iron from 
ferritin is a process with an "all or none" character. 
Electron micrographs of "synthetic ferritin" show that the iron- 
containing micelles in the centres of the molecules have been regene- 
rated to a certain extent. About one half of the apoferritin molecules 
is partly filled up with "iron hydroxide". Complete refilling does 
not take place, probably because of denaturation of the apoferritin 
molecules during removal of the iron. 
4. HEMOCYANIN 
Hemocyanins are oxygen-transporting, copper-containing blood 
proteins occurring freely dissolved in the blood of several inverte- 
brates as crabs, squids, snails and scorpions. The molecular weights 
of the undissociated molecules range from about 800,000 to about 
8,000,000 depending on the biological origin. The molecules can be 
dissociated reversibly into smaller units by changing the p H  or the 
kind and the concentration of the salts present in the solution. 
A survey is given of the literature on hemocyanin. Our own work 
mainly deals with: 
1. The structural relationship between the undissociated mole- 
cules and the dissociation products of Helix pomatia (roman snail) 
and Octopus vulgaris (a squid) hemocyanins. 
2. A possible structural correspondence between the undissociated 
hemocyanin molecules of different biological origin. We compared 
the hemocyanins of Helix pomatia, Octopus vulgaris, Sepia offici- 
na!is (another squid) and Cancer pagurus (a crab). 
Electron micrographs were taken from Helix pomatia hemocyanin 
at different p H  vaiues with the negative-staining method. The disso- 
ciation was studied as a function of p H  at both sides of the isoelectric 
point. Our observations are in agreement with results reported pre- 
viously by other authors, although they are not compatible with the 
model and mechanism of dissociation proposed previously. The 
electron micrographs suggest that the Helix pomatia hemocyanin 
molecule is roughly cylindrical; the diameter of the cylinder is about 
300 A and the height is about 335 A. This cylinder consists of a 
stack of 6 parallel plates of subunits and has a five-fold axis. The 
first dissociation step occurs perpendicular to the five-fold axis into 
halves. Further dissociation is supposed to be a sectorwise splitting 
of these flat cylinders via submultiples into subunits. Divalent cations 
in concentrations of about 1 0 - ~  molar or higher are required for 
the reversibility of the dissociation. 
Preliminary results indicate a similar behaviour for Helix pomatia 
apohemocyanin. 
Electron micrographs of Octopus vulgaris and Sepia officinalis 
hemocyanins show the molecules in the region of the isoglectric point 
as flat cylinders consisting of 3 parallel layers perpendicular to a 
five-fold axis. These cylinders have a height of about 140 A and 
a diameter of about 300 A. They closely resemble the half-molecules 
of Helix pomatia hemocyanin, although, amongst others, differences 
are observed in the arrangement of the subunits. 
The dissociation as a function of p H  was preliminarily studied 
at  both sides of the isoelectric point. The influence of divalent cations 
on this dissociation was not investigated. 
Cancer pagurus hemocyanin consists of smaller molecules, which 
are tentatively represented as a pentalateral prism. 
The undissociated molecules of Helix pomatia, Sepia officinalis, 
Octopus vulgaris and probably those of Cancer pagurus have their 
cylindrical (prismatical) layer structures in common. These layers 
are perpendicular to the cylinder axis, which is an axis of five-fold 
symmetry. 


Elektronenmicroscopie van ferritine en hemocyanine

ARTS repository - University of Groningen

   University of GroningenElektronenmicroscopie van ferritine en hemocyanineBruggen, Ernst Frederik Jacobus vanIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1962Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bruggen, E. F. J. V. (1962). Elektronenmicroscopie van ferritine en hemocyanine. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 09-09-2023Summary ELECTRON MICROSCOPY OF FERRITIN AND HEMOCYANIN 1. INTRODUCTION The concepts primary, secondary, tertiary and quarternary struc- ture of proteins are described. Electron microscopy chiefly deals with the quarternary structure: the way protein molecules are built from subunits. By electron microscopy we have studied the structural changes of ferritin and hemocyanin molecules under the influence of different chemical agents. 2. TECHNIQUES The preparation of carbon films and the way protein molecules are sprayed onto the carbon film are described. The various staining methods for proteins can be divided in shadow-techniques, positive- and negative-staining methods. Several negative-staining agents are discussed e.g. potassium phosphotungstate (PTA), sodium tungstate, uranyl acetate and a complex of uranyl acetate with ethylene diamine tetraacetate ("uranyl. EDTA-complex"). Use was made of the Siemens Elmiskop-I and the Philips EM-100 and EM-200 electron microscopes. The beam voltages were between 40 and 80 kV, the "spot" size was 15-25 ,U, the emission current about 20 pA and the diameter of the objective aperture 25-50 p. The astigmatism of the objective lens was reduced to less then 0.2 ,L[. Photographs were taken on Ilford 5Bll  35 mm film at  a magni- fication of 18,000-35,000 X and on Ilford Special Lantern Plates (gradation: contrasty) at a magnification of 40,000-80,000 X. The films were developed in a ten times diluted Glycinool developer (make: Amaloco), the plates in Kodak D61A developer for 15 and 3 minutes respectively. The developing temperature was 18-20' C. 3. FERRITIN Ferritin is a remarkable protein, because of its high iron content (about 20 Olo of its dry weight). The iron is concentrated in a micelle of composition (FeO.OH),(FeO.OPO,H,). When the iron is removed from ferritin by the action of reducing agents apoferritin is formed. b I t  is possible to introduce some iron again into apoferritin, by which a "synthetic ferritin" is obtained. A short survey is given of the literature on ferritin. I t  was shown previously by electron microscopy that a ferritin molecule consists of the iron containing micelle surrounded by an approximately spherical shell of apoferritin. We studied the structure of the ferritin molecule in some detail, the intermediate stages of the molecules during the removal of the iron, the apoferritin molecule and the molecule of "synthetib fer- ritin". Use was made of the replica and shadow-technique of HALL h and several negative-staining methods. A model is given for the iron-containing micelle; it  consists of 6 subunits at the corners of a trilateral prism (pointgroup symmetry - 6). The structure of apoferritin could not be elucidated, although an indication of the arrangement of the subunits has been obtained. The centre of the apoferritin molecule is hollow and filled with the surrounding solution. In Table I11 the FeIN-weight ratios of ferritin, apoferritin and the intermediate stages are given. The proportions of the number of full, partly full and empty molecules as observed on electron micrographs are listed for each Fe/N-ratio. The results are also shown in Figure I. I t  is concluded that the removal of iron from ferritin is a process with an "all or none" character. Electron micrographs of "synthetic ferritin" show that the iron- containing micelles in the centres of the molecules have been regene- rated to a certain extent. About one half of the apoferritin molecules is partly filled up with "iron hydroxide". Complete refilling does not take place, probably because of denaturation of the apoferritin molecules during removal of the iron. 4. HEMOCYANIN Hemocyanins are oxygen-transporting, copper-containing blood proteins occurring freely dissolved in the blood of several inverte- brates as crabs, squids, snails and scorpions. The molecular weights of the undissociated molecules range from about 800,000 to about 8,000,000 depending on the biological origin. The molecules can be dissociated reversibly into smaller units by changing the p H  or the kind and the concentration of the salts present in the solution. A survey is given of the literature on hemocyanin. Our own work mainly deals with: 1. The structural relationship between the undissociated mole- cules and the dissociation products of Helix pomatia (roman snail) and Octopus vulgaris (a squid) hemocyanins. 2. A possible structural correspondence between the undissociated hemocyanin molecules of different biological origin. We compared the hemocyanins of Helix pomatia, Octopus vulgaris, Sepia offici- na!is (another squid) and Cancer pagurus (a crab). Electron micrographs were taken from Helix pomatia hemocyanin at different p H  vaiues with the negative-staining method. The disso- ciation was studied as a function of p H  at both sides of the isoelectric point. Our observations are in agreement with results reported pre- viously by other authors, although they are not compatible with the model and mechanism of dissociation proposed previously. The electron micrographs suggest that the Helix pomatia hemocyanin molecule is roughly cylindrical; the diameter of the cylinder is about 300 A and the height is about 335 A. This cylinder consists of a stack of 6 parallel plates of subunits and has a five-fold axis. The first dissociation step occurs perpendicular to the five-fold axis into halves. Further dissociation is supposed to be a sectorwise splitting of these flat cylinders via submultiples into subunits. Divalent cations in concentrations of about 1 0 - ~  molar or higher are required for the reversibility of the dissociation. Preliminary results indicate a similar behaviour for Helix pomatia apohemocyanin. Electron micrographs of Octopus vulgaris and Sepia officinalis hemocyanins show the molecules in the region of the isoglectric point as flat cylinders consisting of 3 parallel layers perpendicular to a five-fold axis. These cylinders have a height of about 140 A and a diameter of about 300 A. They closely resemble the half-molecules of Helix pomatia hemocyanin, although, amongst others, differences are observed in the arrangement of the subunits. The dissociation as a function of p H  was preliminarily studied at  both sides of the isoelectric point. The influence of divalent cations on this dissociation was not investigated. Cancer pagurus hemocyanin consists of smaller molecules, which are tentatively represented as a pentalateral prism. The undissociated molecules of Helix pomatia, Sepia officinalis, Octopus vulgaris and probably those of Cancer pagurus have their cylindrical (prismatical) layer structures in common. These layers are perpendicular to the cylinder axis, which is an axis of five-fold symmetry. 

NARCIS 

   University of GroningenElektronenmicroscopie van ferritine en hemocyanineBruggen, Ernst Frederik Jacobus vanIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1962Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bruggen, E. F. J. V. (1962). Elektronenmicroscopie van ferritine en hemocyanine. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 08-06-2022Summary ELECTRON MICROSCOPY OF FERRITIN AND HEMOCYANIN 1. INTRODUCTION The concepts primary, secondary, tertiary and quarternary struc- ture of proteins are described. Electron microscopy chiefly deals with the quarternary structure: the way protein molecules are built from subunits. By electron microscopy we have studied the structural changes of ferritin and hemocyanin molecules under the influence of different chemical agents. 2. TECHNIQUES The preparation of carbon films and the way protein molecules are sprayed onto the carbon film are described. The various staining methods for proteins can be divided in shadow-techniques, positive- and negative-staining methods. Several negative-staining agents are discussed e.g. potassium phosphotungstate (PTA), sodium tungstate, uranyl acetate and a complex of uranyl acetate with ethylene diamine tetraacetate ("uranyl. EDTA-complex"). Use was made of the Siemens Elmiskop-I and the Philips EM-100 and EM-200 electron microscopes. The beam voltages were between 40 and 80 kV, the "spot" size was 15-25 ,U, the emission current about 20 pA and the diameter of the objective aperture 25-50 p. The astigmatism of the objective lens was reduced to less then 0.2 ,L[. Photographs were taken on Ilford 5Bll  35 mm film at  a magni- fication of 18,000-35,000 X and on Ilford Special Lantern Plates (gradation: contrasty) at a magnification of 40,000-80,000 X. The films were developed in a ten times diluted Glycinool developer (make: Amaloco), the plates in Kodak D61A developer for 15 and 3 minutes respectively. The developing temperature was 18-20' C. 3. FERRITIN Ferritin is a remarkable protein, because of its high iron content (about 20 Olo of its dry weight). The iron is concentrated in a micelle of composition (FeO.OH),(FeO.OPO,H,). When the iron is removed from ferritin by the action of reducing agents apoferritin is formed. b I t  is possible to introduce some iron again into apoferritin, by which a "synthetic ferritin" is obtained. A short survey is given of the literature on ferritin. I t  was shown previously by electron microscopy that a ferritin molecule consists of the iron containing micelle surrounded by an approximately spherical shell of apoferritin. We studied the structure of the ferritin molecule in some detail, the intermediate stages of the molecules during the removal of the iron, the apoferritin molecule and the molecule of "synthetib fer- ritin". Use was made of the replica and shadow-technique of HALL h and several negative-staining methods. A model is given for the iron-containing micelle; it  consists of 6 subunits at the corners of a trilateral prism (pointgroup symmetry - 6). The structure of apoferritin could not be elucidated, although an indication of the arrangement of the subunits has been obtained. The centre of the apoferritin molecule is hollow and filled with the surrounding solution. In Table I11 the FeIN-weight ratios of ferritin, apoferritin and the intermediate stages are given. The proportions of the number of full, partly full and empty molecules as observed on electron micrographs are listed for each Fe/N-ratio. The results are also shown in Figure I. I t  is concluded that the removal of iron from ferritin is a process with an "all or none" character. Electron micrographs of "synthetic ferritin" show that the iron- containing micelles in the centres of the molecules have been regene- rated to a certain extent. About one half of the apoferritin molecules is partly filled up with "iron hydroxide". Complete refilling does not take place, probably because of denaturation of the apoferritin molecules during removal of the iron. 4. HEMOCYANIN Hemocyanins are oxygen-transporting, copper-containing blood proteins occurring freely dissolved in the blood of several inverte- brates as crabs, squids, snails and scorpions. The molecular weights of the undissociated molecules range from about 800,000 to about 8,000,000 depending on the biological origin. The molecules can be dissociated reversibly into smaller units by changing the p H  or the kind and the concentration of the salts present in the solution. A survey is given of the literature on hemocyanin. Our own work mainly deals with: 1. The structural relationship between the undissociated mole- cules and the dissociation products of Helix pomatia (roman snail) and Octopus vulgaris (a squid) hemocyanins. 2. A possible structural correspondence between the undissociated hemocyanin molecules of different biological origin. We compared the hemocyanins of Helix pomatia, Octopus vulgaris, Sepia offici- na!is (another squid) and Cancer pagurus (a crab). Electron micrographs were taken from Helix pomatia hemocyanin at different p H  vaiues with the negative-staining method. The disso- ciation was studied as a function of p H  at both sides of the isoelectric point. Our observations are in agreement with results reported pre- viously by other authors, although they are not compatible with the model and mechanism of dissociation proposed previously. The electron micrographs suggest that the Helix pomatia hemocyanin molecule is roughly cylindrical; the diameter of the cylinder is about 300 A and the height is about 335 A. This cylinder consists of a stack of 6 parallel plates of subunits and has a five-fold axis. The first dissociation step occurs perpendicular to the five-fold axis into halves. Further dissociation is supposed to be a sectorwise splitting of these flat cylinders via submultiples into subunits. Divalent cations in concentrations of about 1 0 - ~  molar or higher are required for the reversibility of the dissociation. Preliminary results indicate a similar behaviour for Helix pomatia apohemocyanin. Electron micrographs of Octopus vulgaris and Sepia officinalis hemocyanins show the molecules in the region of the isoglectric point as flat cylinders consisting of 3 parallel layers perpendicular to a five-fold axis. These cylinders have a height of about 140 A and a diameter of about 300 A. They closely resemble the half-molecules of Helix pomatia hemocyanin, although, amongst others, differences are observed in the arrangement of the subunits. The dissociation as a function of p H  was preliminarily studied at  both sides of the isoelectric point. The influence of divalent cations on this dissociation was not investigated. Cancer pagurus hemocyanin consists of smaller molecules, which are tentatively represented as a pentalateral prism. The undissociated molecules of Helix pomatia, Sepia officinalis, Octopus vulgaris and probably those of Cancer pagurus have their cylindrical (prismatical) layer structures in common. These layers are perpendicular to the cylinder axis, which is an axis of five-fold symmetry. 

Het doel van het onderzoek is de bestudering van de verandering van eiwitmolekulen onder invloed van verschillende agentia. Hierbij hebben we ons beperkt tot twee eiwitten, ferritine en hemocyanine.
Dit werk vormt een deel van het onderzoek van de "Werkgemeenschap Eiwitten"van de Stichting "Scheikundig Onderzoek Nederland" en werd mede mogelijk gemaakt door geldelijke steun van de Nederlandse Organisatie voor Zuiver Wetenschappelijk Onderzoek (ZWO) ...

Zie: Summary

Bruggen, Ernst Frederik Jacobus van,

University of Groningen Digital Archive

University of Groningen

   University of GroningenElektronenmicroscopie van ferritine en hemocyanineBruggen, Ernst Frederik Jacobus vanIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1962Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bruggen, E. F. J. V. (1962). Elektronenmicroscopie van ferritine en hemocyanine. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019Summary ELECTRON MICROSCOPY OF FERRITIN AND HEMOCYANIN 1. INTRODUCTION The concepts primary, secondary, tertiary and quarternary struc- ture of proteins are described. Electron microscopy chiefly deals with the quarternary structure: the way protein molecules are built from subunits. By electron microscopy we have studied the structural changes of ferritin and hemocyanin molecules under the influence of different chemical agents. 2. TECHNIQUES The preparation of carbon films and the way protein molecules are sprayed onto the carbon film are described. The various staining methods for proteins can be divided in shadow-techniques, positive- and negative-staining methods. Several negative-staining agents are discussed e.g. potassium phosphotungstate (PTA), sodium tungstate, uranyl acetate and a complex of uranyl acetate with ethylene diamine tetraacetate ("uranyl. EDTA-complex"). Use was made of the Siemens Elmiskop-I and the Philips EM-100 and EM-200 electron microscopes. The beam voltages were between 40 and 80 kV, the "spot" size was 15-25 ,U, the emission current about 20 pA and the diameter of the objective aperture 25-50 p. The astigmatism of the objective lens was reduced to less then 0.2 ,L[. Photographs were taken on Ilford 5Bll  35 mm film at  a magni- fication of 18,000-35,000 X and on Ilford Special Lantern Plates (gradation: contrasty) at a magnification of 40,000-80,000 X. The films were developed in a ten times diluted Glycinool developer (make: Amaloco), the plates in Kodak D61A developer for 15 and 3 minutes respectively. The developing temperature was 18-20' C. 3. FERRITIN Ferritin is a remarkable protein, because of its high iron content (about 20 Olo of its dry weight). The iron is concentrated in a micelle of composition (FeO.OH),(FeO.OPO,H,). When the iron is removed from ferritin by the action of reducing agents apoferritin is formed. b I t  is possible to introduce some iron again into apoferritin, by which a "synthetic ferritin" is obtained. A short survey is given of the literature on ferritin. I t  was shown previously by electron microscopy that a ferritin molecule consists of the iron containing micelle surrounded by an approximately spherical shell of apoferritin. We studied the structure of the ferritin molecule in some detail, the intermediate stages of the molecules during the removal of the iron, the apoferritin molecule and the molecule of "synthetib fer- ritin". Use was made of the replica and shadow-technique of HALL h and several negative-staining methods. A model is given for the iron-containing micelle; it  consists of 6 subunits at the corners of a trilateral prism (pointgroup symmetry - 6). The structure of apoferritin could not be elucidated, although an indication of the arrangement of the subunits has been obtained. The centre of the apoferritin molecule is hollow and filled with the surrounding solution. In Table I11 the FeIN-weight ratios of ferritin, apoferritin and the intermediate stages are given. The proportions of the number of full, partly full and empty molecules as observed on electron micrographs are listed for each Fe/N-ratio. The results are also shown in Figure I. I t  is concluded that the removal of iron from ferritin is a process with an "all or none" character. Electron micrographs of "synthetic ferritin" show that the iron- containing micelles in the centres of the molecules have been regene- rated to a certain extent. About one half of the apoferritin molecules is partly filled up with "iron hydroxide". Complete refilling does not take place, probably because of denaturation of the apoferritin molecules during removal of the iron. 4. HEMOCYANIN Hemocyanins are oxygen-transporting, copper-containing blood proteins occurring freely dissolved in the blood of several inverte- brates as crabs, squids, snails and scorpions. The molecular weights of the undissociated molecules range from about 800,000 to about 8,000,000 depending on the biological origin. The molecules can be dissociated reversibly into smaller units by changing the p H  or the kind and the concentration of the salts present in the solution. A survey is given of the literature on hemocyanin. Our own work mainly deals with: 1. The structural relationship between the undissociated mole- cules and the dissociation products of Helix pomatia (roman snail) and Octopus vulgaris (a squid) hemocyanins. 2. A possible structural correspondence between the undissociated hemocyanin molecules of different biological origin. We compared the hemocyanins of Helix pomatia, Octopus vulgaris, Sepia offici- na!is (another squid) and Cancer pagurus (a crab). Electron micrographs were taken from Helix pomatia hemocyanin at different p H  vaiues with the negative-staining method. The disso- ciation was studied as a function of p H  at both sides of the isoelectric point. Our observations are in agreement with results reported pre- viously by other authors, although they are not compatible with the model and mechanism of dissociation proposed previously. The electron micrographs suggest that the Helix pomatia hemocyanin molecule is roughly cylindrical; the diameter of the cylinder is about 300 A and the height is about 335 A. This cylinder consists of a stack of 6 parallel plates of subunits and has a five-fold axis. The first dissociation step occurs perpendicular to the five-fold axis into halves. Further dissociation is supposed to be a sectorwise splitting of these flat cylinders via submultiples into subunits. Divalent cations in concentrations of about 1 0 - ~  molar or higher are required for the reversibility of the dissociation. Preliminary results indicate a similar behaviour for Helix pomatia apohemocyanin. Electron micrographs of Octopus vulgaris and Sepia officinalis hemocyanins show the molecules in the region of the isoglectric point as flat cylinders consisting of 3 parallel layers perpendicular to a five-fold axis. These cylinders have a height of about 140 A and a diameter of about 300 A. They closely resemble the half-molecules of Helix pomatia hemocyanin, although, amongst others, differences are observed in the arrangement of the subunits. The dissociation as a function of p H  was preliminarily studied at  both sides of the isoelectric point. The influence of divalent cations on this dissociation was not investigated. Cancer pagurus hemocyanin consists of smaller molecules, which are tentatively represented as a pentalateral prism. The undissociated molecules of Helix pomatia, Sepia officinalis, Octopus vulgaris and probably those of Cancer pagurus have their cylindrical (prismatical) layer structures in common. These layers are perpendicular to the cylinder axis, which is an axis of five-fold symmetry. 

Elektronenmicroscopie van ferritine en hemocyanine

Abstract

Similar works

Full text

Available Versions

University of Groningen Research Database

ARTS repository - University of Groningen

NARCIS

ARTS repository - University of Groningen

University of Groningen Digital Archive

University of Groningen