Interaction of Proteins with Nanostructured Latex Particles in Aqueous Solution A. Wittemann, B. Haupt, University of Bayreuth E. Breininger, T. Neumann, M. Rastätter, N. Dingenouts, University of Karlsruhe T. Narayanan, ESRF, Grenoble
Problem 1: Immobilization of enzymes on nanoparticles Colloidal particles large surface large amount of immobilized protein/enzyme Enzymes can be used as catalysts for technical applications
Goal 2: Avoiding adsorption of proteins on surfaces Adsorption on solid surface Denaturation of proteins by adsorption on solid surfaces strong attraction by van der Waals or hydrophobic interaction M. Santore et al., Langmuir 2002, 18 (3), 706. Remedied by attached polymers through steric repulsion?
Interaction of polyelectrolyte brushes with dissolved proteins Charged Brush; no added salt solid substrate Counterions confined within brush chains strongly stretched Protein/charged brush strong repulsion expected solid substrate Proteins are attracted to polyelectrolyte brush!
Carrier particles: spherical polyelectrolyte brush CH2 CH COO Annealed brush R CH2 CH SO 3 Quenched brush L Can be used as carrier particles for enzymes Long charged macromolecules attached chemically to colloidal particles
CH2 CH COO Menu: R CH2 CH SO 3 L 1. Spherical polyelectrolyte brushes Protein solution Mixing Ultrafiltration Protein coated brush latex dissolved proteins Protein coated brush latex Brush latex Glucoamylase substrate 2. Adsorption of protein on spherical polyelectrolyte brushes product 3. Use as nanoreactors
PhotoEmulsionpolymerization Synthesis of Spherical Polyelectrolyte Brushes Photoinitiator O C O O 2 O C OH O O O C.. C OH C O Polymerization of watersoluble monomers on surface Generation of two radicals growth of chains on surface and in serum free chains removed by serum replacement Guo, Ballauff, Langmuir 2000, 16, 8719
Polyacrylic acid attached to surface of particles: Dependence of radius on ph 300 1M 0.1M 0.01M 0.001M 0.0001M annealed brush /PAA 200 L [nm] 100 0 0 2 4 6 8 10 12 14 ph Guo, MB; Phys. Rev. E. 64, 051406 (2001) Parameter: concentration of added salt Strong swelling due to confined counterions
MDSimulation of spherical polyelectrolyte brushes confinement of counterions Arben Jusufi 140 chains with 10 monomers Each monomer charged large fraction of counterions confined
CryoTEM imaging of Spherical Polyelectrolyte brushes (SPB) seeing is believing CH 2 CH SO 3 R L 200nm Markus Drechsler, Yeshayahu Talmon, Alexander Wittemann
low ionic strength (7mM) osmotic brush high ionic strength (107mM) salted brush 100nm 100nm
Spherical Polyelectrolyte Brush Confinement of counterions R L confined counterions: High osmotic pressure inside brush layer Determines properties of particles in solution
Experimental procedure: A. Wittemann Phys. Chem. Chem. Phys. 2003, 5, 1671 ph fixed through MESbuffer Protein solution Mixing Ultrafiltration Ionic strength adjusted by monovalent salt Brush latex Protein coated brush latex dissolved proteins Protein coated brush latex Ultrafiltration after 24 h: A certain part of protein remains affixed to brush layer
adsorption of BSA in polyelectrolyte brush: Ionic strength decisive parameter τ ads [mg/g SPB] 1200 800 400 10mM MES 10mM MES 25mM NaCl 10mM MES 50mM NaCl 10mM MES 100mM NaCl 10mM MES 150mM NaCl Osmotic brush Marked adsorption of BSA at low ionic strength 0 0 4 8 12 c sol [mg/ml] Salted brush adsorption suppressed in presence of salt Phys. Chem. Chem. Phys. 2003, 5, 1671
Influence of ph τ ads [mg/g SPB] 2000 1600 1200 800 400 0 0 4 8 12 ph 5,1 ph 6,1 ph 7,2 ph: important but not decisive parameter Adsorption takes place on wrong side above IP c sol [mg/ml] Phys. Chem. Chem. Phys. 2003, 5, 1671
Comparison with conventional latexes: 1200 adsorption on spherical polyelectrolyte brush τ ads [mg/g SPB] 800 400 0 0 2 4 6 8 c sol [mg/ml] M. Rastätter, A. Wittemann multilayer formation in brush layer adsorption on "conventional" carboxylated polystyrene latex (5 wt% acrylic acid) monolayer adsorption on surface
driving force: release of counterions of negative chains and positive patches blue: red: basic residues acidic residues yellow: neutral residues Positive patches become counterions for polyelectrolyte chains of brush solid substrate
Interaction of proteins with brush layer: counterion release force main driving force (Wittemann et al. 2003) High osmotic pressure partially relieved by multivalent counterions Solid substrate () Solid substrate () Counterion release force: established concept from biophysics (Rädler et al. ; v. Grünberg et al.)
Smallangle Xray scattering (SAXS): Location of proteins within brush layer
SAXS: Localisation of adsorbed proteins 1000 60 I(q) [cm 1 ] 10 0.1 ρ(r) [nm 3 ] 40 20 0 0 20 40 60 80100 r [nm] 0.001 0 0.2 0.4 0.6 0.8 q [nm] BSA enters into brush layer Rosenfeldt et al., Phys. Rev. E 2004, 70, 061403
SAXS: Localisation of adsorbed proteins Rnase A brush RNase A bare brush Rosenfeldt et al., Phys. Rev. E 2004, 70, 061403 RNase A enters into brush layer except region near to surface
Secondary structure analysis of proteins embedded in SPB Amid I band: coupled C=Ostretch vibration random coil H α Helix N υ O β sheet conformation sensitive!
Secondary structure analysis of proteins embedded in SPB arbitrary absorbance units 1.0 0.8 0.6 0.4 0.2 0 1800 Analyt. Chem. 2004, 76, 2813 initial BSA BSA adsorbed BSA desorbed 1600 1400 wave number [cm 1 ] 1200 FTIR: No significant change in spectra Secondary structure of is retained during adsorption to SPB
Activity of bound enzymes substrate Glucoamylase Glucoamylase product starch Assay: glucoamylase glucose glucoamylase 2chloro4nitrophenol 2chloro4 nitrophenolmaltotrioside Assay: Measurement of absorption at 405nm
Glucoamylase substrate product Activity of bound glucoamylase: MichaelisMenten analysis v 0 = v max K [ S ] [ S ] M 1/v [Lmin/mmol] 3000 2000 1000 free enzyme 28 mg/g CP 97 mg/g CP 160 mg/g CP 232 mg/g CP Activity of enzyme preserved 0 0 0.5 1.0 1.5 1/ [S] [L/mmol] Macromol. Biosci. 2004, 4, 13; Biomacromolecules, in press
Nanostructured latex particles: Conclusions R CH2 CH2 CH COO CH SO 3 protein Carrier particles for proteins L substrate Glucoamylase Confinement of counterions Nanoreactor product
Acknowledgement Alexander Wittemann Systems, isotherms, FTIR Enzyme kinetics: B. Haupt SAXS: E. Breininger, S. Rosenfeldt, N. Dingenouts, T. Narayanan (ESRF) TEM: M. Drechsler, Prof. Y. Talmon (Technion, Haifa) University of Dortmund C. Czeslik, G. Jackler University of Ulm Prof. U. Nienhaus, C. Röcker, K. Anikin : DFG, BMBF, Roche Diagnostics, BASF AG, Fonds der Chemischen Industrie