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

SAXS: Localisation of proteins within brush layer

Comparison with conventional latexes

Wittemann et al., Z. Phys. Chem. NF, in press.

SAXS beamline ID 2, ESRF Grenoble

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

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