Investigation of biopolymer-mineral interactions in the natural composite material nacre [Elektronische Ressource] / von Fabian David Heinemann

Noelte

Description

Investigation of Biopolymer - MineralInteractions in the Natural CompositeMaterial NacreVom Fachbereich fur¨ Physik und Elektrotechnikder Universit¨at Bremenzur Erlangung des akademischen Grades einesDoktor der Naturwissenschaften (Dr. rer. nat.)genehmigte Dissertationvon Dipl. Phys. Fabian David Heinemannaus Lohfelden / Kassel1. Gutachterin: Prof. Dr. Monika Fritz2. Gutachter: Prof. Dr. Dr. h.c. Horst A. DiehlEingereicht am: 24.11.2008Tag des Promotionskolloquiums: 19.12.2008Contents1. Summary 52. Introduction 72.1. Motivation ..................................... 72.2. Abalone Nacre ................................... 82.2.1. Shell Growth ................................122.2.2. Nacre Growth132.3. Targets of the Thesis . ...............................182.3.1. Crystallization Templated by the Interlamellar Matrix .........182.3.2. Determining the Surface Free Energy of the Interlamellar Matrix . . . 182.3.3. Influence of the Soluble Matrix on Precipitation Rates182.3.4. Amino Acid Analysis............................193. Material and Methods 213.1. Organic Material Extraction from Nacre .....................23.1.1. Nacre Preparation from Abalone Shells .................223.1.2. Preparation of the Interlamellar Organic Matrix ............23.1.3. Deproteination of the In Organic Matrix...........223.1.4. Preparation of the Water Soluble Matrix233.2. Chemical Methods . . ...............................243.2.1.

Sujets

• Biologie

Informations

• Publié par: universitat_bremen
• Publié le: 01 janvier 2008
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 19 Mo

Extrait

InvestigationofBiopolymer-Mineral

InteractionsintheNaturalComposite

1.

2.

NacreMaterial

VomFachbereichf¨urPhysikundElektrotechnik
Bremenat¨Universitder

zurErlangungdesakademischenGradeseines

DoktorderNaturwissenschaften(Dr.rer.
Dissertationgenehmigte

vonDipl.Phys.FabianDavidHeinemann
Kassel/Lohfeldenaus

Gutachterin:Prof.Dr.MonikaFritz

Gutachter:Prof.Dr.Dr.h.c.HorstA.Diehl

24.11.2008am:Eingereicht

TagdesPromotionskolloquiums:19.12.2008

nat.)

Contents

5rySumma1.7ductionIntro2.7.....................................ationMotiv2.1.8...................................NacreAbalone2.2.2.2.1.ShellGrowth................................12
2.2.2.NacreGrowth................................13
2.3.TargetsoftheThesis................................18
2.3.1.CrystallizationTemplatedbytheInterlamellarMatrix.........18
2.3.2.DeterminingtheSurfaceFreeEnergyoftheInterlamellarMatrix...18
2.3.3.InfluenceoftheSolubleMatrixonPrecipitationRates.........18
2.3.4.AminoAcidAnalysis............................19
3.MaterialandMethods21
3.1.OrganicMaterialExtractionfromNacre.....................22
3.1.1.NacrePreparationfromAbaloneShells.................22
3.1.2.PreparationoftheInterlamellarOrganicMatrix............22
3.1.3.DeproteinationoftheInterlamellarOrganicMatrix...........22
3.1.4.PreparationoftheWaterSolubleOrganicMatrix............23
3.2.ChemicalMethods.................................24
3.2.1.ProteinConcentrationDetermination(BradfordAssay)........24
3.2.2.CreationofPolymerFoils.........................25
3.3.CharacterizationMethodsforCrystallizationProducts.............26
3.3.1.X-RayDiffraction.............................26
3.3.2.ScanningElectronMicroscopy.......................27
3.3.3.AragoniteSensitiveStaining(FeiglTest).................28
3.4.Double-DiffusionCrystallizationExperiments..................29
3.4.1.Introduction................................29
3.4.2.ExperimentalProcedure..........................29
3.5.ContactAngleMeasurements...........................31
3.5.1.Introduction................................31
3.5.2.TheoreticalBackground..........................31

2

Contents

3

3.5.3.ExperimentalProcedure..........................35
3.6.CaCO3PrecipitationAssay............................37
3.6.1.Introduction................................37
3.6.2.ExperimentalProcedure..........................37
3.7.AminoAcidAnalysis................................39
3.7.1.Introduction................................39
3.7.2.TheoreticalandMethodicalBackground.................39
3.7.3.ExperimentalProcedures.........................43
47DiscussionandResults4.4.1.Double-DiffusionCrystallizationunderConstantConditions..........48
4.1.1.Introduction................................48
4.1.2.Ammonium-CarbonateMethod......................48
4.1.3.ConventionalDouble-Diffusion......................49
4.1.4.DevelopmentofaCrystallizationDeviceforControlledCrystallization
onaPolymericSurface...........................50
4.1.5.ResultsandDiscussion...........................52
4.2.SurfaceFreeEnergyDetermination........................60
4.2.1.Introduction................................60
4.2.2.ResultsandDiscussion...........................60
4.2.3.HypothesisofFlatCrystalGrowthinNacreduetoSurfaceFreeEn-
ergyProperties...............................63
4.3.CaCO3PrecipitationAssay............................65
4.3.1.Introduction................................65
4.3.2.DevelopmentofaCalculationMethodforCaCO3PrecipitationRates65
76...................................Results4.3.3.07..................................Discussion4.3.4.4.4.AminoAcidAnalysis................................74
4.4.1.Introduction................................74
4.4.2.EstablishmentandCalibrationoftheMethod..............74
4.4.3.ValidationoftheProcedureUsingKnownProteins...........76
4.4.4.SolubleOrganicMatrixofNacre.....................78
5.ConclusionandPerspectives84
87Bibliography96endixAppA.A.1.DeterminationoftheOrganicFraction......................97
A.2.WorkforHeterogeneousandHomogeneousNucleation.............99
A.3.Double-DiffusionBox................................101

Contents

A.4.

A.5.

A.6.

A.7.

A.8.

A.9.

Self-MadeTurbiditySensor............................

............................CalibrationyAssaBradford

Abbreviations....................................

...............................ufacturersManofList

.................................tEquipmenA.7.1.

ChemicalsA.7.2........................Consumablesand

..................................publicationsOwn

Acknowledgements/Danksagungen

.......................

4

601

701

801

190

901

901

101

111

1rySumma

Theiridescentandhighlyfractureresistantnaturalcompositematerialnacreconsistsof
plateletsofaragonite(aCaCO3polymorph),arrangedinahighlyregularmanner.Anor-
ganicmatrixoflessthan5%inweightisintercalatedbetweenthemineralplatelets,whichis
necessaryfortheself-organizedgrowthandthemechanicalpropertiesofnacre.Inthisthe-
sis,aspectsoftheself-organizedgrowthaswellaspropertiesoftheorganicmatrixobtained
fromnacreofthemarinegastropodHaliotislaevigata(greenlipabalone)wereinvestigated.

Theinfluenceoftheinterlamellarmatrix(thewaterinsolublematrixcomponent)on
CaCO3crystalnucleationandgrowthwasinvestigated.Adoublediffusiondevicewithcon-
stantrenewalofthesolutionswasdeveloped,suitableforthecrystallizationunderconstant
conditions.Bycrystallizationontheinterlamellarmatrixandinvestigationwithscanning
electronmicroscopyandX-raydiffraction,itwaspossibletoshowthattheinterlamellar
matrixofnacrenucleatesflataragonitecrystalswithamorphologystronglyresemblingthe
aragonitecrystalsinnacre.Incontrolexperimentsonseveralpolymerfoils,onlytheusual
crystalmorphologiesandnoselectivityforthepolymorpharagonitewasobserved.
Foramoredetailedcharacterizationoftheinterlamellarmatrix,thesurfacefreeenergy
wasdeterminedusingcontactanglemeasurementswiththesessiledropmethod.Thesemi-
empiricalevaluationmethodofOwensandWendtwasused,allowingthedeterminationof
surfacefreeenergycomponentsarisingfromdispersiveandpolarinteractions.Thesurface
freeenergyoftheinterlamellarmatrixismainlyofnon-polarorigin.Itcouldbeshownby
proteindigestionthatproteinsonthesurfaceoftheinterlamellarmatrixareresponsiblefor
thisbehavior.Inaddition,ahypothesiswasdeveloped,thattheunusualcrystallizationof

5

rySumma1.

6

flataragonitecrystalsisaresultofsuitablesurfacefreeenergypropertiesoftheinterlamellar
matrix,favoringnucleationandwettingbyaragonite.
Sincealsothesolublematrixisrequiredfornacregrowth,theinfluenceofsolublematrix
proteinsonCaCO3precipitationrateswasdetermined.Asimpleprecipitationassaywas
usedasabasisandextended.ThepH-valuecourseandinitialconditionswereusedto
calculatetherateofCaCO3crystallization.Thesolubleproteinsfromnacreshowedahigh
influenceonCaCO3precipitationratescomparedtotheproteinsbovineserumalbuminand
lysozyme.Atconcentrationsofmorethan1.0μg/ml,thesolublematrixproteinswerestrong
crystalgrowthinhibitors,whereasatlowconcentrationsaround0.04μg/ml,crystallization
enhancementofafactorof1.5wasobserved.
Thesolublematrixwasfurthercharacterizedbyperforminganaminoacidanalysisof
thesolublematrixproteins.Afterdemineralizationand0.22μmfiltration,thematrix
wasanalyzedeitheras-isorpurifiedbycation-exchangepurification,C18-ZipTipbind-
ingorultracentrifugation.Aminoacidanalysiswasperformedbyhighperformanceliquid
chromatographicseparationofphenylthiocarbamylaminoacidsofthehydrolyzedsamples.
Withoutpurification,theamountofnon-proteinmatterwastoohighforaminoacidanal-
ysis.Theaminoacidcompositionwasstronglydependentonthemethodofmatrixprotein
purification.Ingeneral,proline,glycine,leucineandalaninewerethemostdominantamino
acids.ApronouncedcontentofasparticacidwasfoundonlyafterC18-ZipTippurification.
Indicatorsforacollagen-likecomponentwerefound:highprolineandglycinecontents,as
wellasthepossibledetectionofhydroxyproline.

Motivation2.1.

2Introduction

Inthelivingworld,awidevarietyoforganismsiscapableofutilizingthedepositionof
minerals.Thisabilitydevelopedveryearlyinthehistoryoflifeandsubsequentlyevolved,
beginningwiththesimplebiologicallyinducedbiomineralization,theprecipitationofmin-
erals,indistinguishablefromgeologicalprecipitates[Lowenstam,1981],tothebiologically
controlledbiomineralization[Mann,1983],theformationofcomplexandhighlyspecialized
compositematerialslikebone,enamelornacre.
Probablytheoldestfossilrecordsoflife-datingbackmorethan3.5billionyears[Allwood
etal.,2007]-arestromatolites,chalkstonestructuresinthesea,coveredbymicroorganisms.
Theformationofthesestructuresisinducedbythemicroorganisms,whichinitiatecalcium
carbonateprecipitationfromtheseawater.
However,themuchmorecomplexmaterialscreatedbybiologicallycontrolledbiominer-
alizationrequireaveryhighdegreeofcontrolovercrystalnucleationandgrowth.Sofar,
theformationprocessesofallcomplexbiomineralsareonlypoorlyunderstood.

Severalpropertiesofnacremakeitanespeciallyvaluableobjectofresearchinthefield
ofbiomineralization.Sinceithasahighlyorderedandcomparativelysimplestructure
withonlytwohierarchicallevels,itmayserveasamodelsystemforbiomineralization.
Principlesthatexplaintheprocessesofnacreformationmayturnouttobegeneralprinciples
ofbiomineralization.Adeeperknowledgeofcalciumcarbon