chapter8 protein interactions with biomaterials 8.1 introduction: thermodynamics of protein...
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CHAPTER
88Protein Interactionswith Biomaterials
8.1 Introduction: Thermodynamics of Protein Adsorption
Biomaterial surface + Protein adsorption --- Body reaction Protein adsorption properties ---- Biocompatibility 결정
Composition of proteins and their binding effects on the material surfaces
8.1.1. Gibbs Free Energy and Protein Adsorption Protein (P) to an empty site on a surface (*): P + * = P*
Gads = Hads – T Sads
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individual reaction before and after the protein adsorption 1) for the protein: Gprot = Hprot – T Sprot
2) for the solvent near the adsorption surface: Gsol = Hsol – T Ssol 3) for the biomaterial surface: Gsurf = Hsurf – T Ssurf
therefore, Gads = Gprot + Gsol + Gsurf
8.1.2. Systems Properties Governing Protein Adsorption surface hydrophobicity and charge distribution factors influencing protein adsorption:
1) dehydration of the surface and protein2) redistribution of charged groups3) structural rearrangement of the protein
Interactions between domains on the biomaterial surface & the protein
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(1) dehydration for protein adsorptionhydrophobic interaction ---- water disorder ---- entropy 증가
(2) charge interaction surface --- protein repulsion --- counter ions --- energy expenditure
(3) structural rearrangement of the protein less stable protein --- preferable adsorption (conformational rearrangement)
8.2. Protein Structure8.2.1. Amino Acid Chemistry
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8.2.2. Primary Structure DNA --- RNA --- Protein
codon usagemissense mutation [HbA & HbS]
8.2.3. Secondary Structure -helix -sheets [parallel vs. antiparallel]
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8.2.4. Tertiary Structure TIM-barrel fold (triosephosphate isomerase) Noncovalent interactions
ionic interactions / hydrogen bonding / hydrophobic interaction / van der Waals
Locations of hydrophobic and hydrophilic residues
8.2.5. Quaternary Structure subunits --- regulatory (allosteric) enzymes
8.3. Protein Transport and Adsorption Kinetics [prior to the adsorption]
8.3.1. Transport to the Surface four types: diffusion / thermal convection / flow / coupled transport
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parabolic velocity profile ex) blood in the vessel
when r = R, V = 0---- no flow at the surface
convective and diffusional contributions to overall protein transport
protein transport through a solution + protein adsorption to the surface with boundary condition
rate of protein adsorption at the surface = rate of transport of protein to the surface
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8.3.2. Adsorption Kinetics biomaterial at the wall --- protein adsorption via diffusion alone
high initial adsorption rate and plateau value
protein rearrangement on the surfaceprotein monolayer
protein rearrangement to improve packing
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8.4. Reversibility of Protein Adsorption 8.4.1. Reversible & Irreversible Binding 1) short period of reversible binding
initial contact of protein --- conformation change [protein unfolding and spreading on the material]
2) irreversible (permanent) binding no desorption [simultaneous desorption]
3) final protein conformation --- activity
8.4.2. Desorption and Exchange no desorption, but protein exchange
[dynamic nature of protein attachment] protein displacement
final protein composition on the surface--- conc. of protein & surface affinity
(size, charge, hydrophobicity, structural stability, unfolding rate)
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diffusion-dependent adsorption --- eventual replacement with greater surface affinity [Vroman effect for protein exchange]
8.5. Techniques: Assays for Protein Type and Amount 8.5.1. High-Performance Liquid Chromatography (HPLC):
Affinity Chromatography (1) Basic principles
adsorption chromatography normal-phase: polar stationary phase + nonpolar mobile phasereversed-phase: nonpolar stationary phase + polar mobile phase
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(2) Instrumentation detector: UV, refractive index, fluorescence(3) Information provided area: quantitative analysis with calibration curve fractionation (non-destructive monitoring)
8.5.2. Colorimetric Assays (1) Basic principles and Instrumentation marker chemical + protein --- chromophore 형성
UV/VIS spectrometry detection (Beer-Lambert’s Law) product from substrate by enzyme
absorbance monitoring for assessing enzyme activity
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8.5.3. Fluorescent Assays (1) Basic principles
fluorophore + protein --- fluorescence (radiationless decay)
(2) Instrumentation (3) Information provided protein quantitation
sensitivity intrinsic fluorescence (trp, tyr, phe)
8.5.4. Enzyme-linked Immunosorbent Assay (ELISA) sandwich ELISA
surface - primary Ab – protein – secondary Ab – enzyme (alkaline phosphatase, horseradish peroxidase, p-nitrophenol phosphatase)
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8.5.5. Western Blotting (1) Basic principles and procedures
SDS-PAGE ---- protein transfer to nitrocellulose paper