Biofunctionalization by spontaneous adsorption of proteins K. Henzler, A. Wittemann, S. Rosenfeldt,...
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Transcript of Biofunctionalization by spontaneous adsorption of proteins K. Henzler, A. Wittemann, S. Rosenfeldt,...
Biofunctionalization by spontaneous adsorption of proteins
K. Henzler, A. Wittemann, S. Rosenfeldt, B.
Haupt, M. Ballauff
University of Bayreuth
Aim: Immobilization of proteins on colloidal carriers
„bionanoparticles“Colloidal particles
• provide large surfaces
• large amount of immobilized biomolecules
Enzymes can be used as catalysts for technical applications
substrate
bound enzymes
product
Problem: Adsorption on solid surfaces has to be avoided!
M. Santore et al., Langmuir 2002, 18 (3), 706.
Adsorption on solid surface
Denaturation of proteins by adsorption on solid surfaces
- strong attraction by van der Waals or hydrophobic interaction
Loss of biological function
PS
R
L
CHCH2
COO-
CHCH2
SO3-
• Long charged polyelectrolytes attached to colloidal particles
weak polyelectrolyte
strong polyelectrolyte
Can be used as carrier particles for proteins
Spherical Polyelectrolyte Brush (SPB)
Confinement of counterions inside brush layer
PS
R
L
CHCH 2
SO 3-
Confined counterions
• high osmotic pressure inside brush
• chains strongly stretched
Properties of the particles determined by the confinement of the counterions
+
negatively charged
negatively charged
carrier protein
Adsorption on the „wrong side“: pH > pI
Double trouble: Electrostatic repulsion + steric repulsion
? ?
Protein adsorption on Spherical Polyelectrolyte Brushes ?
Protein adsorption: Experimental procedure
Wittemann et al., Phys. Chem. Chem. Phys. 2003, 5, 1671.
Ultra-
filtration
PS
PSPS
MixingProtein solution
Brush latex
Protein coated brush latex
Protein coated brush latex + dissolved proteins
A certain amount of protein remains adsorbed after exhaustive ultrafiltration!
Osmotic brush
• high adsorption
Salted brush
• adsorption suppressed
Wittemann et al., Phys. Chem. Chem. Phys. 2003, 5, 1671.
Adsorption of BSA: Parameter ionic strength
Confocal microscopy (K. Anikin, C. Röcker, U. Nienhaus, Ulm)
Adsorption of a fluorescent protein
Anikin et al., Phys. Chem. B 2005, 109, 5418.
• SPB adsorbed on PEG modified surface
• washing with salt solution (250 mM)
• washing with protein solution
Main driving force: Counterion release force
Uptake of protein leads to release of counterions
• strong driving force for protein adsorption even at „wrong“ side of the IEP
High osmotic pressure partially relieved by multi-valent counterions
++
+
+---
+
+
+--
--
-
---
-+
+
+
+
+
+
+++
----
N+ N-
--
-+
+
+
--
--
-
---
-+
+
+
+
+
+++
+
++++
----
2N+ - N- released counterions
++++
++
++------
+
+
+--
--
-
---
-+
+
+
+
+
+++
++
++----
----
--
------
--++
++
++
++
++
++
+++
----
N+ N-
----
--++
++
++
----
----
--
------
--++
++
++
++
++
++++
+
++++
----++++
++
+++++
----+++
----
2N+ - N- released counterions
Polyelectrolyte Mediated Protein Adsorption (PMPA)
Review on the PMPA:
Wittemann, A.; Ballauff, M. Phys. Chem. Chem. Phys. 2006.
Theoretical description:
Leermakers, F.A.M.; Ballauff, M.; Borisov, O.V. Langmuir, in press.
Analysis of the bioconjugates
1. Analysis of the SPB + other carrier systems through scattering methods and electron microscopy
2. Mapping of the adsorbed biomolecules: Cryo-TEM, SAXS
3. Kinetics of the adsorption: TR-SAXS
4. Biofunctionality: Secondary structure analysis,
enzymatic activity
Dynamic Light Scattering (DLS)
Parameter: concentration of added salt
L: height of brush on particle
Guo et al., Phys. Rev. E 2001, 64,051406.
CH2 CH
COOH
low ionic strength
high ionic strength
Wittemann et al., J. Am. Chem. Soc. 2005, 127, 9688.
Cryogenic transmission electron microscopy (Cryo-TEM)
Osmotic brush: confined counterions, cs > ca
Salted brush: cs = ca
Localisation of adsorbed BSA
Rosenfeldt et al., Phys. Rev. E 2004, 70,061403.
brush +BSA
brush
BSA in solution
BSA enters into brush layer
Localisation of adsorbed protein cont‘d
Ribonulease A
Bovine hemoglobin
Adsorption onto SPB consisting of strong polyelectrolytes
SAXS Beamline ID 2, ESRF / Grenoble; local contact: T. Narayanan
Adsorption kinetics: Time-resolved SAXS
PS
PSMixing
Protein solution
Brush latex
Protein coated brush latex + dissolved proteins
Secondary structure analysis in turbid media
• Protein signal recorded in suspension
Wittemann et al., Anal. Chem. 2004, 76, 2813.
• Substraction of the spectra of the SPB
Parameter:amount of adsorbed BSA
BSA before adsorption
BSA adsorbed
reliberated BSA
Wittemann et al., Anal. Chem. 2004, 76, 2813.
Amide-I-band
Amide-II-band
Retention of the native secondary structure
Secondary structure analysis in turbid media cont‘d
starch
glucoamylase -D-glucose
2-chloro-4-nitrophenol
2-chloro-4-nitrophenol-maltotrioside
Measurement of absorption at 405 nm
Assay:
Activity of bound enzymes
glucoamylase
e.g. glucoamylase
Activity of enzyme preserved
SK
Svv
M max0
Activity of bound glucoamylase: Michaelis-Menten analysis
Neumann et al., Macromol. Biosci. 2004, 4, 13; Haupt et al., Biomacromolecules 2005, 6, 948.
-1/Km
Isothermal Titration calorimetry (ITC)
R S
T1 = 0
dt
dQ
HVv
app
10
0 1 2 3
0.0
0.1
0.2
0.3
UV-Vis ITC
[S]
v0 KM, kcat
time [min]
P [m
ol/
s]
UV/VISITCalternative solution to
determine KM, kcat
1. Optimization of the PMPA; viability for biofunctionalization
2. Other carrier systems (bottle brushes, polyelectrolyte stars, biodegradable nanoparticles)
3. Synthesis of biofunctionalized nanoparticles for diagnostics and drug delivery
Perspectives in the framework of BIOSONS
PS
Aim: „Nanoplant“
Cascade reactions: Possible system:
-Amylase:
starch maltose
-Glucosidase:
maltose glucose
Glucose Oxidase:
glucose H2O2enzyme A enzyme B
end product
PS
R
L
CHCH2
COO-
CHCH2
SO3- protein
„Nanoreactor“
Carrier particles for proteins
Confined counterions
Conclusions
Location of proteins within brush layer by SAXS: Model calculations
Rosenfeldt et al., Phys. Rev. E 2004, 70, 061403
Low q: adsorbed protein leads to core-shell structure
10-16
10-15
10-14
10-13
10-12
0 0.1 0.2 0.3 0.4
intensity of small spheresstrawberry core-shell particleCore-shell particle + small spherescore particle
q [nm-1
]
I 0(q)
[cm
2 ]
Location of proteins within brush layer by SAXS: Model calculations cont‘d:
High q: adding of intensities 10-17
10-16
10-15
10-14
0.4 0.8 1.2
core-shell
protein
strawberry
q [nm-1]
I 0(q)
[cm
2 ]
SAXS: Monitoring the adsorption of BSA
0.001
0.1
10
1000
0 0.2 0.4 0.6q [nm]
I(q)
[cm
-1]
020
4060
80
0 20 40 60 80 100r [nm]
(r
) [n
m-3
]
unloaded
296mg/g
1116mg/g
Rosenfeldt et al., Phys. Rev. E 2004, 70, 061403
Proteins adsorbed onto conventional latexes: no desorption
Protein is liberated when salt concentration inside layer salt conc. outside
Release of adsorbed protein when ionic strength is raised
Wittemann et al., Z. Phys. Chem. NF, in press.
Adsorption of BSA: influence of pH
Wittemann et al., Phys. Chem. Chem. Phys. 2003, 5, 1671.
pH important but not as decisive as ionic strength
Adsorption takes place on „wrong side“ above IEP
ca
cs
pHa
pHs
Wittemann et al., Phys. Chem. Chem. Phys. 2003, 5, 1671.
Biesheuvel et al., J. Phys. Chem. B 2005, 109, 4209.
Protein near IEP: Charge reversal by lower pH within brush
Lower pH because of confined protons
-protonation of NH2 groups on surface
Charge reversal cannot be the only reason for the strong adsorption!
But: Glucoamylase (pI = 3.5) is adsorbed at pH = 6.1
Neumann et al., Macromol. Biosci. 2004, 4, 13; Haupt et al., Biomacromolecules 2005, 6, 948.
blue: basic residues
red: acidic residues
yellow: neutral residues
Protein surface: „patches“ of positive and negative charge
Positive patches become counterions for polyelectrolyte chains of SPB
-
--
-
-
--
-
- -
++++ -
-
--
- -
main driving force given by Donnan-pressure D
Difference between salt concentrations inside and outside creates Donnan-pressure
Counterion release force
Wittemann et al., Phys. Chem. Chem. Phys., in press; Progr. Colloid Polym. Sci. 2006, 133, 58.
+
+
+--
--
-
---
-+
+
+
+
+
+
ca
cs
+-
+-
Direct correlation between strength of adsorption and D
Résumé
Polyelectrolyte-protein complexes
Polyelectrolyte-mediated protein adsorption
• both can take place on the „wrong side“ of the IEP
• both can be traced back to patches of opposite chargeHowever: PMPA leads to stronger protein binding because of the Donnan effect and a much stronger correlation of the counterions
Synthesis: photoemulsion polymerization
Guo et al., Macromolecules 1999, 32, 6043.
r.t., h
acrylic acid
PS
PAA
PS
photo-initiator
70°CPS
Step 1:
PS latex
Step 2
Photoinitiator layer
Step 3
Shell composed of linear polyelectrolytes
Synthesis of SPB: „grafting from“ technique
CO
O
OHOC
O
PS
C
O CO
O
OOH. .+
h
PS
• photo-initiator decomposed into two radicals
growth of chains on surface and in serum
• free chains removed by serum replacement
Polymerization of water-soluble monomers on latex particles
SPB: decisive parameters
R: core radius
L: hydrodynamic brush layer thickness
LC: contour length of the poly(acrylic acid)
chains
: grafting density of the tethered chains
D: average distance of the grafting points
PS
PAA
R
LC
D
L
Guo et al., Langmuir 2000, 16, 8719.
Secondary structure of adsorbed proteins
Amide-I-band: coupled C=O-stretch vibration
N
O
H
υ
conformation sensitive
FT-IR spectroscopyrandom coil
- helix
- sheet
wave number [cm-1]
wave number [cm-1]
wave number [cm-1]
-helix
-sheet RNase A in sol.
Temperature-induced unfolding of RNase A
RNase A
-helix
-sheet adsorbedRNase A
Wittemann et al., Macromol. Biosci. 2005, 5, 13.
Proteins
• BSA
• RNase A
• BLG
• Glucoamylase
• -, -Glucosidase
• FP
• hemoglobin
• myoglobin
• amylase
Different proteins – different adsorption strength
BSA
BLG
Wittemann et al., Anal. Chem. 2004, 76, 2813.
Rosenfeldt et al., Phys. Rev. E 2004, 70,061403.
Localisation of adsorbed proteins
brush +RNase A
bare brush
Counterion release force
Donnan-pressure D within brush a measure for the adsorption
Difference between salt concentration inside and outside create Donnan-pressure
Direct correlation between strength of adsorption and D
2-nitrophenyl--D-glucopyranoside
Substrate:
Hydrolysis of 1,4- and 1,6--glycosidic bonds with release of -D-glucose
Activity of bound -glucosidase
Haupt et al., Biomacromolecules 2005, 6, 948.
id
kTnRid
Osmotic coefficient : Fraction of „free“ counterions
Measurement of the osmotic pressure of dilute salt-free suspensions
ca. 95 % of counterions confined in brush as predicted for osmotic limit
water
membrane
p < 0
pressure
solution
Acknowledgement
University of Bayreuth Prof. M. Ballauff
Enzyme kinetics: B. Haupt
SAXS: K. Henzler, E. Breininger,
S. Rosenfeldt, N. Dingenouts
TEM: M. Schrinner, M. Drechsler, Prof. Y. Talmon
(Technion, Haifa)
Pau / St. Petersburg O.V. Borisov
Wageningen University Prof. F.A.M. Leermakers
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