8 Protein Interactions with Biomaterials CHAPTER 8.1 Introduction: Thermodynamics of Protein Adsorption Bacterial 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 for the protein: Gprot = Hprot – T Sprot for the solvent near the adsorption surface: Gsol = Hsol – T Ssol 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 protein 2) redistribution of charged groups 3) structural rearrangement of the protein Interactions between domains on the biomaterial surface & the protein

dehydration for protein adsorption hydrophobic 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 Structure 8.2.1. Amino Acid Chemistry

8.2.2. Primary Structure DNA --- RNA --- Protein codon usage missense mutation [HbA & HbS] 8.2.3. Secondary Structure a-helix b-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 surface protein 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 Basic principles adsorption chromatography normal-phase: polar stationary phase + nonpolar mobile phase reversed-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 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 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 Basic principles and procedures SDS-PAGE ---- protein transfer to nitrocellulose paper