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

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

(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

8.2.2. Primary Structure DNA --- RNA --- Protein

codon usagemissense mutation [HbA & HbS]

8.2.3. Secondary Structure -helix -sheets [parallel vs. antiparallel]

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

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

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

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)

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

(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

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)

8.5.5. Western Blotting (1) Basic principles and procedures

SDS-PAGE ---- protein transfer to nitrocellulose paper