Transcript Characterization of Proteins and Nucleic Acids on Biomaterials

Characterization of Protein- &
Biomolecule-based
Biointerfaces
Prof. Prabhas Moghe
November 13, 2006
125:583
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Outline
• Methods for assessing concentration of biomolecules
– ELISA
– Radiolabeling
– Fluorometry
• Methods for assessing thickness, conformation,
organization of biomolecules at interfaces
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Circular Dichroism
FTIR Spectroscopy (Reviewed)
Atomic force microscopy (Reviewed)
Ellipsometry
Total Internal Reflection Fluorescence (TIRF) (Reviewed)
• Methods for estimating protein or ligand affinity to
interface
– Scatchard Analysis (Radiolabeling)
– Surface Plasmon Resonance (SPR) (Reviewed)
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Direct Determination Using Labeled
Species
• Need to label
• Usually need to remove solution from
material
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FTIR vs. Radiolabeleing
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Data analysis: the Langmuir isotherm
Cs
Cbulk

• Basic equation: fb 
Cs,max K  Cbulk
• Assumptions:
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Distinct adsorption sites
Single type (binding energy) of site
Independence of sites
Solute doesn’t change form after adsorption
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Determining Amounts of Biomolecules on
Surfaces: Preliminaries
• Useful properties of biomolecules
(absorbance; fluorescence; birefringence)
• Labeling (use of fluorophore or radiolabeling)
• Typical amounts
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What is an ELISA?
• Enzyme-linked immunosorbent assay
• Name suggests three components
– Antibody (immuno)
• Allows for specific detection of analyte of interest
– Solid phase (sorbent)
• Allows one to wash away all the material that is not
specifically captured
– Enzymatic amplification
• Allows you to turn a little capture into a visible color change
that can be quantified using an absorbance plate reader
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Capture and Detection Antibodies
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Sandwich ELISA
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Competitive ELISA
• Less is more. More antigen in your sample
will mean more antibody competed away,
which will lead to less signal
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Enzymes with Chromogenic Substrates
• High molar extinction coefficient (i.e., strong
color change)
• Strong binding between enzyme and
substrate (low KM)
• Linear relationship between color intensity
and [enzyme]
v
kcat  E   S 
K M   S 
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If you’re lucky…
Sample Standard Curve
0.5
Aborbance (490 nm)
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
y = -0.0583Ln(x) + 0.3858
R2 = 0.9919
Log. (absorbance)
absorbance
0.05
0
0.1
1
10
100
Concentration (ug/mL)
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X-ray photoelectron spectroscopy (aka
ESCA)
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Paper # 1
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RGD peptide immobilization
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Ellipsometry
Polarization:
Equipment Schematic:
Measure:

Rp
Rs

Rp


 exp  i  p   s 


Rs
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Ellipsometry
•
Ellipsometry consists of the measurement of the change in polarization
state of a beam of light upon reflection from the sample of interest. The
exact nature of the polarization change is determined by the sample's
properties (thickness and refractive index). The experimental data are
usually expressed as two parameters Y and D. The polarization state of
the light incident upon the sample may be decomposed into an s and a
p component (the s-component is oscillating parallel to the sample
surface, and the p-component is oscillating parallel to the plane of
incidence). The intensity of the s and p component, after reflection, are
denoted by Rs and Rp. The fundamental equation of ellipsometry is
then written:
RP

 tan  eiD , where
Rs
tan  is amplitude change upon reflection; D is phase shift
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Ellipsometry through Substrate Layers
(Advanced review)
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Back to RGD peptide example
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Determining Characteristics of Adsorbed
Biomolecular Structure
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Single molecule protein detection approaches
Piehler, COSB, 15:4-14, 2005
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Circular dichroism (CD)
Dichroism is the phenomenon in which light absorption differs for different directions of
polarization. Linear dichroism involves linearly polarized light where the electric vector is
confined to a plane. Circularly polarized light contrasts from linearly polarized light. In
linearly polarized light the direction of the electric vector is constant and its magnitude is
modulated; in circularly polarized light the magnitude
is constant and the direction is modulated, as shown below.
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Circular Dichroism Principle
The differential absorption of radiation polarized in two directions as function of frequency
is called dichroism. When applied to plane polarized light, this is called linear dichroism; for circularly
polarized light, circular dichroism. We can think of linear polarized light as the result of two equal
amplitudes of opposite circular polarization. After passing through an optically active sample, circularly
polarized light will be changed in two aspects. The two components are still circularly-polarized, but
the magnitudes of the counter-rotating E-components will no longer be equal as the molar extinction
coefficients for right- and left-polarized light are unequal. The direction of the E-vector no longer traces
a circle - instead it traces an ellipse. There will also be a rotation of the major axis of the ellipse due to
differences in refractive indices.
The optics for making circularly polarized
light uses a linear polarizer P and a
quarter-wave retarder R. Circularly
polarized light can be decomposed in the
sum of two mutually perpendicular
linearly polarized waves that are one
quarter of a wavelength out of phase.
With Ey retarded on quarter of a wave
relative to Ez, we have right circularly
polarized light as diagrammed here. If Ez
were retarded one quarter of a wave
relative to Ey, then the circularly
polarized light would be left-handed.
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CD (continued)
• 2 related properties
– Optical rotation: differential
transmission of circularly
polarized light in left- and
right-hand directions
– Circular dichroism:
differential absorption of
left- and right-circularly
polarized light
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Circular dichroism spectroscopy is particularly
good for:
* determining whether a protein is folded, and if so characterizing its secondary structure,
tertiary structure, and the structural family to which it belongs
* studying the conformational stability of a protein under stress -- thermal stability, pH
stability, and stability to denaturants -- and how this stability is altered by buffer
composition or addition of stabilizers and excipients
* determining whether protein-protein interactions alter the conformation of protein. Small
conformational changes have been seen, for example, upon formation of several
different receptor/ligand complexes.
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Secondary structure can be determined by CD spectroscopy in the far-uv
spectral region (190-250 nm). At these wavelengths the chromophore is the
peptide bond, and the signal arises when it is located in a regular, folded
environment. Alpha-helix, beta-sheet, and random coil structures each give
rise to a characteristic shape and magnitude of CD spectrum. The
approximate fraction of each secondary structure type that is present in any
protein can thus be determined by analyzing its far-uv CD spectrum as a
sum of fractional multiples of such reference spectra for each structural
type.
Like all spectroscopic techniques, the CD signal reflects an average of
the entire molecular population. Thus, while CD can determine that a
protein contains about 50% alpha-helix, it cannot determine which specific
residues are involved in the alpha-helical portion.
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CD of proteins and nucleic acids
Nucleic acids:
Proteins:
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CD of adsorbed T4 lysozyme
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Characterizing Interactions of
Biomolecules on Surfaces
Estimation of ligand affinity to substrates
Ligand-Receptor Binding
Ligand-Cell Binding
Ligand-Substrate Binding (Paper)
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Binding of labeled ligands to receptors
Concentration of monovalent ligand, L (moles/volume or M)
Concentration of monovalent receptor, R (#/cell)
Concentration of Complex, C (#/cell)
Concentration of cells=n (#/unit volume)
kf
R  LÉ C
kr
RT  R(t)  C(t)
Lo  L(t)  (
n
).C(t)
N Av
If ligand depletion is ignored,
At equilibrium,
dC(t)
 k f R(t).L(t).  kr C(t)
dt


 n 
dC(t)
 k f RT  C(t) Lo  
.C(t)
  kr C(t)

dt
N
 AV 


dC(t)
 k f RT  C(t)Lo  kr C(t)
dt
 k f Lo RT 
C(t)  Co exp k f Lo  kr  
 1  exp  k f Lo  kr t 
k
L

k
 f o
r




k f  RT  Ceq gLo  kr Ceq
RT Lo
kr
Ceq 
; KD 
K D  Lo
kf
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Binding of labeled ligands to receptors,
continued
• Let u be the scaled/dimensionless number of
ligand-receptor complexes, =C/RT
ueq
u
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Scatchard Plot to estimate KD
 RT 
1

Ceq  
L
KD
 K D 
Ceq
Bound ligand
 1 / K D .(Bound ligand)  (RT / K D )
Free ligand
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Paper # 2:
Affinity of biomolecular binding to
substrates
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Experimental System
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Adsorption Model
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Kinetic Model for Binding
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Fitting the best model to experimental data
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