Week_2_-_SPLecture_2_1
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### Week 2 - SPLecture 2_1: Protein Affinity vs Protein Activity
Lecturer: Prof. Karin Hing
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Page 1: Introduction
- Topic: Biocompatibility - Protein Affinity vs Protein Activity
- Recap from Week 1:
- Protein shape is controlled by primary structure and local environment.
- Protein shape regulates its function.
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Page 2: Key Points from Week 1
- Protein adsorption occurs whenever a material is introduced into a biological environment.
- The protein interlayer is influenced by:
- Material properties (physio-chemistry)
- Protein characteristics (structure, chemistry)
- Host environment (composition)
- Key factors behind protein interlayer formation:
- Dehydration & hydrophobicity
- Electrostatics & surface charge
- Conformational changes & protein stabilisation
- Importance of protein interlayers in bioactivity:
- The host interacts with the material through the adsorbed protein layer.
- If the material does not release substances, this interaction becomes the primary biological response driver.
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Page 3: Observed Cell Responses to Materials
- Osteogenic cell adhesion and activity are usually superior on hydrophilic surfaces.
- Example: cp titanium & hydroxyapatite outperform polyethylene.
- Many resorbable polymers are naturally hydrophobic and require surface modification before cell seeding.
- Example: Polystyrene in tissue culture plastics is modified to become hydrophilic to improve cell attachment.
- The protein interlayer quality impacts cell response based on:
- What proteins are present (affinity).
- How proteins are arranged (structure and activity).
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Page 4: Role of the Protein Interlayer in Cell Response
- Protein interlayer quality depends on:
- Affinity/Enrichment (which proteins adsorb preferentially)
- Activity/Activation (whether proteins retain function upon adsorption)
- Affinity/Enrichment:
- Determines how specific proteins adsorb to a material or tissue surface in a given environment.
- Activity/Activation:
- A protein’s ability to function biologically after adsorption, which depends on its conformation and ability to interact with other molecules.
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Page 5: Protein Affinity
- Protein affinity depends on the environment:
- Vitronectin adsorbs equally to hydrophilic and hydrophobic surfaces in pure solutions.
- However, in serum, hydrophilic surfaces adsorb more vitronectin, improving cell attachment and migration.
- Dilution experiments can help identify affinity-driven enrichment.
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Page 6: Environmental Sensitivity of Affinity
- Factors affecting protein affinity:
- Temperature & protein concentration:
- Adsorption levels vary at 18°C vs 37°C.
- Solute composition:
- The presence of different media (MEM, PBS, HA, SA) alters adsorption levels.
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Page 7: Competitive Adsorption
- Co-adsorbants affect protein binding:
- Presence of BSA and whole serum changes adsorption behaviour.
- Activity also matters:
- Same amount of fibronectin adsorbed, but cell attachment rate varies depending on surface pre-conditioning.
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Page 8: Key Protein Characteristics Affecting Affinity & Activity
- Properties influencing protein behaviour at surfaces:
- Composition & Conformation
- Solubility & Hydrophobicity
- Molecular weight
- Charge & Isoelectric point (pI)
- Stability
- Protein stability affects function:
- Tertiary structure influences how a protein behaves when adsorbed.
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Page 9: Protein Molecular Weight & Isoelectric Point (pI)
- Examples of protein molecular weights:
- Fibronectin: 440,000 – 480,000 Da
- Vitronectin: 65,000 – 75,000 Da
- Hemoglobin: 64,500 Da
- Isoelectric point (pI) determines protein charge at a given pH.
- Proteins are less soluble near their pI.
- At pH 7.4, fibronectin and vitronectin carry a net negative charge.
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Page 10-11: Protein Stability
- Proteins are only marginally stable in physiological conditions (ΔG ~ 20-65 kJ/mol).
- Denaturation can occur due to:
- Heating
- Extreme pH
- Chaotropic agents
- Surface adsorption
- Impact of stability on activity:
- A protein’s functionality depends on its structural integrity.
- Orientation & conformation impact biological activity.
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Page 12: Protein Activity & Surface Activation
- Protein function changes upon adsorption:
- Fibronectin and vitronectin may lose or gain function depending on how they adsorb.
- Example: Fibronectin shows reduced cell-binding ability in its soluble form, requiring surface activation.
- Vitronectin partially unfolds upon binding, improving integrin binding.
- Fibrinogen binds to inactivated platelets only when adsorbed.
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Page 13: Modulating Surface Activation
- Competitive adsorption can alter protein function:
- In high-protein environments, adsorption-induced conformational changes can be suppressed.
- Fibronectin on hydrophilic polystyrene supports better osteoblast adhesion than on hydrophobic polystyrene.
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Page 14: Hydrophilic vs Hydrophobic Surfaces
- Cell adhesion and proliferation:
- More successful on moderately hydrophilic surfaces than hydrophobic ones.
- Fibronectin must reorganise after adsorption to support cell function.
- Hydrophobic surfaces limit reorganisation, reducing function.
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Page 15: Enhanced Protein Activity on Hydrophilic Surfaces
- Cell response variation is linked to:
- Surface charge
- Hydrophobicity
- Point of zero charge
- Higher activity on silicate-substituted HA (SA) compared to hydroxyapatite (HA).
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Page 16: Role of Specific Binding Sites
- Protein orientation matters:
- Specifically charged surfaces can control protein orientation.
- Example: Osteopontin’s cell-binding ability is modulated by surface charge.
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Page 17: Summary – Key Factors Influencing Protein Affinity & Activity
- Material characteristics:
- Composition
- Surface chemistry
- Wettability
- Surface charge
- Stability
- Protein characteristics:
- Structure
- Conformation
- Solubility
- Charge & pI
- Molecular weight
- Environmental conditions:
- Ionic & organic composition
- Temperature
- pH, pO₂, pCO₂
Final Key Takeaway:
- Both protein enrichment (affinity) and activation (activity) are crucial for optimising biomaterial-cell interactions.
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### Final Notes