st lecture interfacial protein adsorption and inactivation

							          Chapter 4
interfacial protein adsorption
       and inactivation
                  I introduction
 Protein adsorption phenomena examples
 (1) clinical implants
 (2) mammalian and bacterial cell adhesion to surfaces
 (3) initiation of blood coagulation
 (4) solid-phase immunoassay
 (5) protein binding to cell surfaces
 Protein-enzyme interfacial interaction in bioseparation
 (1) gas-liquid
 (2) liquid-liquid
 (3) liquid-solid
                I introduction
 Protein  interfacial adsorption
 Low protein concentration (no barrier )
 High surface protein concentration (barrier)
 A electrostatic
 B steric
 C osmotic effect
      Proteins adsorption on Gas-liquid
                  interface
   (1) enzyme adsorbed unfolded partially active or inactive state
       as rigid film
   (2) proteins unfolding depends on conformational stability
   (3) interfaces are primarily responsible for protein inactivation
   (4) Shear associated damage is severe when gas-liquid
        interface present
    A pump
    B centrifuges
    C ultrafiltration
   (5) proteins on adsorption at fluid interfaces undergo a change
       from their globular configuration to an extended chain
       structure
   (6) change of protein structure lead to activity changes
                 Proteins adsorption
   1 protein molecules in aqueous solution have a hydrophobic
     interior and a hydrophilic exterior
   2 structural perturbation-rearrangements involving changes in
      interatomic distance affect the intermolecular van der waals
      interaction
   3 charge groups in the apolar interior of the protein molecule
      tend to form ion pairs.
   4 intramolecular peptide hydrogen bonds are more favorable
      than peptide-water interaction
   5 the variations in the hydrogen bonding, ion pairing,
      hydrophobic interaction affect the rotational motion
                Proteins adsorption
   6 Increase of entropy originating from the dehydration of the
      interface-surface, and dehydration of structural changes
      are the driving force.
   7 pH and shaking significantly affect the rate of transport of
      protein to interface.
   8 Surfactants occupy the surface and prevent the unfolding and
      denaturation of protein.
   9 Proteins adsorbed exist multiple binding mode states
   A weakly and tightly bound proteins
   B Not all of adsorbed protein is removed under one or a
      specified set of conditions
    II Reaction and inactivation at liquid-
               liquid interfaces
1   lipid-water interfaces
    A Inhibitory protein prevents the lipase from
         binding
    B Serum albumin and β-lactoglobulin result
        in lipase activity loss of 93 and 92%
    C Different conformational states exhibit
       different activities and activities of this
       protein could be regulated.
      B Aqueous two-phase systems
   (1) Moderate selectivity
   (2) high yield
   (3) Biocompatibility
   (4) amenability to scale-up
   (5) Electrostatic interaction play a significant part in protein
       adsorption and interaction at the interface
   Example 4.1 PEG-FeSO4-water, PEG-Na2SO4-water
   (1) PEG-salt system are attractive compared with PEG-dextran
       system
   (2) PEG concentration level is low in salt-rich phase
       A recovery of biomolecules is easier from salt-rich phase
         using ultrafiltration
       B limited PEG loss
              Reversed micelles
 (1) Surfactant aggregate in organic solvent
 (2) Protein can be induced to move from bulk
     aqueous phase into micelle-containing organic
     solvent and vice versa by manipulation of pH, ionic
     strength, and surfactant concentration.
 (3) Protein transfer is dominated by electrostatic
     interactions
 (4) Selectively separating protein
       Example 4.2 interfacial transport
                 processes
   （1） Transfer of α-chymotrypsin and cytochrome C
   （2） pH and salt concentration influence both forward and
       back transfer rate of protein
   （3） Electrostatics play a significant role
   （4） Charge interaction will produce a significant electrostatic
       force at interface.
   （5） Protein forward transfer faster three orders of magnitude
       than does forward transfer of small molecules
   （6） Extraction kinetics is controlled by convective transport
    Example 4.2 interfacial transport
              processes
Mechanism suggested:
 1 Rupture of organic solvent film between
    reversed micelle and the interface
 2 Reversed micelle may discharge its contents
   to aqueous phase by
   A osmotic pressure difference (for water)
   B coalescence 聚合
   III Reaction and inactivation at gas-
              liquid interfaces
 (1) Adsorption of proteins is important for theory
     and practice
 (2) Protein films assist in stability of foams and
     emulsions
 (3) Transport of dissolved gases in fermentation
     broth
 (4) Foam fractionation and froth flotation
               A shear and agitation
   (1) stirring introduce air into a protein solution
   (2) enzymes with sulfhydryl (SH) group easily
        inactivated by oxidation of disulfides
   (3) inactivation of human hemoglobin A and S
       at air-water interface
      A Hemoglobin A : normal human
      B Hemoglobin S :
         a sickle red cell disease
         b precipitated when shaking
         c shaking induces bubble formation, agitation
           without bubble formation
         d greater area per molecule result in a greater unfolding (S
           8000 A2, A 5000A2
Air-liquid surface effect play major role
          in protein inactivation
   1 Inactivation phenomena
    (1) unfolded
    (2) aggregate
    (3) precipitated
   2 Shaking effect
    (1) Film forms creates a barrier to diffusion without shaking
    (2) Precipitated film material is removed from the interface,
        more protein to be adsorbed and precipitated with shaking
   3 Shear rate plays an important role in inactivation of protein at
     the air-water interface.
   4 Enzyme lost activity due to denaturation of their catalytic sites
     by mechanical shear
Example 4.3 kinetics and mechanism of
     shear inactivation of lipase
   Shearing experiment
   1 Good mixing is essential, but mixing produces shear
   2 Lipase inactivation involved in shear-induced interface effect
     (1) shear increase rate of adsorption at air-water interface
     (2) splits up or change its conformation, unfold, or coagulate.
     (3) shear assist in interface inactivation by replacing old
         molecules at interface and in turn inactivated by
         interface tension
    3 polypropylene glycol (PPG antifoam agent) significantly
       decrease lipase denaturation
     A binds directly to lipase to protect its structure from
       inactivation
     B decreases the surface tension and protects lipase from
       inactivation
Example 4.3 kinetics and mechanism of
     shear inactivation of lipase
4   denaturation rate can be reduced to very
     low level by addition of small amount of
    surfactants or PEG or methylcellulose
 5 denatured β-lactoglobulin can be renatured
   by dissolving it in dilute acid
 6 surfactant preferentially adsorbed occupy the
   surface and prevent the unfolding and
   denaturation.
B techniques for protein adsorption
   Techniques to study adsorption of protein at interfaces
   (1) film balance
   (2) pendant drop methods
   (3) surface tension reduction
   (4) ellipsometry 椭圆光度法
    (5) radiotracer method
     A directly quantify protein concentration at gas-liquid interface
     B more radiation amount from interface than bulk solution
     C high specific and sensitive
     D does not disturb adsorbing protein solution
     E radiolabeling could alter conformation or hydrophobicity of
        molecules
     F common method of radiolabeling is accetylation of terminal amine and
        lysine residues
     G removes charge from derivatized residues and increase hydrophobicity of
        protein
Example 4.4 radiotracer technique
   adsorption of reductively methylated lysozyme to air-water interface
   (1) protein was radiolableled by reductive methylation of terminal amine and
       lysine residues
   (2) obtain an adsorption isotherm for lysozyme measured over a range
       spanning six orders of magnitude of bulk protein concentration
   (3) Adsorption mechanism
      A below a critical concentration: orientation, until saturated monolayer
      B at the critical concentration, end-on orientation
      C above critical concentration, saturated monolayer is
        attached at interface, second layer or multilayer occurs
   (4) isotherm exhibits monolayer saturation at low bulk protein concentration
   (5) lysozyme molecule in first layer do not exchange significantly with
       lysozyme in bulk solution
         Kinetic model developed
   Below critical concentration of protein in bulk
    solution
C model for protein adsorption
              Model explanation
 1 unsuccessful for some proteins such as β-
 lactoglobulin, α-lactalbumin, and BSA
 2 some modification have been suggested
 3 model is simplicity, but still obtain precise and more
    extensive data.
 4 more complex models two adsorption layers
 A first layer at interface
 B second layer from adsorption of protein onto the
    first layer
 C good fit for adsorption of β-casein, BSA, and
    lysozyme
    Peptides adsorption at interfaces
 (1) peptide adsorption is important
      A liquid-solid
      B air-water-liquid
 (2) peptide-hormone drugs(low doses make influence
     of adsorption significant
 (3) using peptides as model substance for adsorption
     studies is important
    A AVP (arginine vasopressin 加压素）and d-
    AVP (desamino-8-arginine vasopressin)
 (4) more such studies are urgently required due to
      medical relevance
    New words and vocabularies
 Implants 植入
 α- helix
 Enthalpy 热焓
 Entropy 熵
 Patches 片
 Perturbation 动摇，混乱
 Ion pairs 离子对
 Plethora 过剩，过多
 Formidable 艰难的，可怕的
 Formate dehydrogenase 甲酸脱氢酶
    New words and vocabularies
 Acrylic 丙烯酸的
 Fall off 下降，跌落
 Cytochrome C 细胞色素C
 Coalescence 合并，联合
 Merge 吞没，融合
 Coagulate 凝结
 Polypropylene glycol PPG
 pendant 悬挂，下垂
 Integer 整数
 Temporal 时间的
                 D Foam fractionation
   Example 4.5
   (1)Foam: made up of gas bubbles dispersed in liquid
   (2) Foam stabilized by surface-active agents
   (3) protein are surfactant and adsorbed on interface of liquid-solid
   (4) parameters affecting foaming behavior
      A pH
      B temperature
      C salts
      D sugars
      E lipids
      F column height
      G column diameter
   (5) conditions to produced or collapse foam are mild
 IV Reaction and inactivation at liquid-
            solid interface
 Protein  adsorption at solid-liquid interface
 (1) biofouling
 (2) thrombosis
 (3) immunologic reaction on solid support
 (4) chromatography separation
 (5) cell culture
 (6) protein drug delivery contact with polymer
      materials
        A Quantitative aspects and
          conformational change
 (1)   quantitative aspects of protein adsorption
 (2)   protein conformation and orientation in
       adsorbed layer
 (3)   mechanisms involved at the solid-liquid
       interface
 (4)   composition and conformational change at
       interface
 (5)   proteins can be displaced by other proteins
       with different characteristics
          A Quantitative aspects and
            conformational change
   (6) protein adsorption depends on both protein and surface
       A intrinsic protein adsorption kinetics
       B chemical equilibrium
       C flow of solution past the adsorbing surface
   (7) absence of techniques for determining the fine three-
       dimensional structure
   (8) driving force for adsorption mainly atributed to entropic
       gains
   (9) minimize protein adsorption by modifying polymer particles
   (10) characterize the conformation of protein adsorbed-
       desorbed from polymer particles
Example 4.6 conformational change of
 proteins adsorbed on small particles
 (1) Proteins adsorption and their conformation
      change determining biological process-interface
      reaction when polymeric material in contact with
      biological fluid
 (2) protein adsorption on polystyrene particles can be
      modulated by coating particles
 (3) proteins unfolded when adsorbed on untreated
      polystyrene particles
 (4) direct evidence for conformational change
 (5) resistant protein adsorption materials may be
     developed
Example 4.7 influence of surface hydrophobicity on the
conformational changes of adsorbed fibrinogen
   Kinetic and adsorption amount of fibrinogen
   (1) Study method
       A fourier transform infrared spectroscopy and attenuated total
         reflection (FTIR-ATR)
       B total internal reflection fluorescence (TIRF)
   (2) Study material
      A germanium
      B poly-hydroxylmethacrylate
      C polystyrene
      D silica
   (3) Results
      A extent of conformational changes is related to surface
        hydrophobicity
      B adsorption induces conformational changes
B adsorption-desorption kinetics theory
   (1) kinetics control the dynamic behavior of molecules at
       interface.
   (2) kinetics types analysis importance
      A proteins adsorption-desorption
      B adsorption of synthetic polymer on colloidal particles
      C manufacturing self assembly monolayers or multilayers
      D relaxation of foams (composed of surfactant-stabilized
        bubbles)
   (3) attractiveness (how quickly molecules in solution are
       adsorbed on surface
   (4) retentiveness duration surface holds onto the adsorbed
       molecule prior to desorption
B adsorption-desorption kinetics theory
 (5) optimize surface-bulk system in terms of weak or
     strong systems to help minimize denaruration of
     protein
 (6) different properites of proteins affect their
      adsorption at solid-liquid interface
    A molecule size
    B charge
    C hydrophobicity of protein
    D flexibility of proteins
 (7) structural adaptability of proteins naturally affect
    their adsorption behavior
    Example 4.8 adsorption behavior of
            different proteins
 1 Proteins material
 A Ribonuclease A
 B Cytochrome C
 C Lysozyme
 D α- lactalbumin
 E BSA
 2 Polymer materials
 A Polystyrene
 B Styrene-2-hydroxyethyl methacrylate
 C Silica
 D Lateral interaction
  Example 4.8 adsorption behavior of
          different proteins
3  Conditions
 A pH
 B Ionic strength
 4 Proteins properties
 A Flexibility
 B Hydrophobicities
 C Isoelectric points
 D Lateral interaction
                   Results
1  hydrophobic interaction for protein-
   hydrophobic surface interaction are stronger
   than electricstatic repulsion
 2 electricstatic interaction between protein-
   hydrophilic surface dominate
 3 larger protein molecules show maxmiun
   adsorption around isoelectric point
 4 proteins interaction affected by a number of
   factors
Example 4.9 Driving forces of savinase
  adsorbed on solid-liquid interface
  Four set of interaction involved in proteins adsorption
 1 Protein-surface interaction (net charge of surface and protein
    is major interaction
 2 Interface dehydration ( dehydration of hydrophobic interface
    promotes protein adsorption, hydrophilic interface opposes it)
   3 a hydrophobic surface and electrostatic repulsion promotes unfolding and
     leads to higher surface area contact of protein
   4 lateral interaction is resisted by electrostatic repulsion
Driving forces of savinase adsorbed on polystyrene and on glass
    A electrostatic interactions are the major driving forces solid-water interface
    B dehydration at hydrophobic interface and lateral interaction are small
       contribution
    C the enzyme adsorbs in its native state, without unfolding
     Adsorption of lysozyme and α-
     lactolbumin at interface
   1 Polymer material
     A Negatively charged polystyrene (PS-)
     B Variably charged hematite (α-Fe2O3)
   2 Force and subprocess of interface adsorption
     A Subprocesses leading to an increase in entropy provide a
        major driving force for protein adsorption
     B Dehydration of sorbent and protein surface is major
        entropy contributions
     C Both lysozyme and α-lactolbumin exhibited a significant
       amount of denaturation on PS-
     D α-lactolbumin is almost completely denatured on
       hydrophilic α-Fe2O3
     E Lysozyme loses only a fraction of its activity due to high
       structure stability
    Example 4.10 Adsorption of fungal
    lipase lipolase at solid-liquid interface
   1 Lipolase extracellular lipase from thermophilic fungus
     Usage: remove fatty soil from clothes in detergents
   2 Lipase: catalyze the hydrolysis and formation of ester bonds
   3 Activity significantly increase at an interface
    A high concentration of substrate
    B a better orientation and conformational change
    C surface active reaction products formed at interface also
      influence subsequent binding
    D delicate balance between surface hydration and
      electricstatic interactions determines adsorption of lipase
      on solid-liquid interface
    E lateral enzyme-enzyme repulsion reaction influences the
     plateau value Lipolase adsorbs as a hard protein, not
     unfolding
          V Conclusions (1)
 1 It is necessary to understand proteins adsorption at
    different types of interface (G-L, L-L, L-S)
 2 Proteins have a thermodymamic tendency to diffuse
 and react at the interface.
 3 Significant and subtle rearrangement will be happen
 as proteins adsorb and react at interface
 4 Adsorbed molecule may continuously change its
 configuration-conformation at interface
          V Conclusions (2)
 5 Most techniques provide quantitative estimate
 without qualitative nature
 6 It is need more realistic and appropriate models for
 proteins adsorption and reaction
 7 Reactions and adsorption of proteins at interface
 should be emphasis
 A Biomedical
 B Immunologic
 C Biotechnological
 D Environmental
 E Other application
    New words and vocabularies (1)
 Drainage 排出
 Collapses 崩溃
 Feedstock 给料
 Conserve 保存，保藏
 Biofouling 生物污垢
 Thrombosis 血栓症
 Prostheses 弥补，修补
 Intervening 干预，介入，干涉
 Mediate 介导
 Fibrinogen 纤维蛋白原
 Kininogen 激肽原
 Cardiovascular 心血管的
    New words and vocabularies (2)
 Clot 凝结
 Antithrombogenicity 抗凝血酶原性
 Concomitant 伴随的
 Heterogeneity 异种性，不同成分
 Nuclear magnetic resonance 核磁共振
 Photon 光子
 Anisotropy 各向异性
 Fluorescence 荧光
 Segmental 部分的
 Attenuate 削弱
 Germanium 锗
 New words and vocabularies (3)
 Poly-(hydroxylmethacrylate)   聚羟甲基丙烯酸
  酯
 Attractiveness 引力
 Retentiveness 保留力
 Surmount 超越，克服
 Adiabatic 绝热
 Lateral 横向
 Savinase 双子柏酶
 Latex 乳胶，橡胶
 Hematite 赤铁矿
    New words and vocabularies (4)
 Dehydration 脱水
 Microcalorimetry 微热测定仪
 Cohesion 内聚力，结合
 Lipolase 脂糖酶
 Delicate 精巧的，灵敏的
 Plateau 高原，高地
 Lipolytic 脂肪分解的
 Ex situ 离位
 In situ   在位
 Manifestation 显示，表现
 Depletion 损耗
 Coordinate 坐标
                            Questions
   1 What change will be happen proteins activity when it adsorbed on
     interface ?
   2 Why aqueous two-phase systems can preserve protein activity well?
   3 Why shear and agitation reduce protein stability?
   4 Why can foams be used for separation of proteins from solution?
   5 Which driving forces at interface adsorption of proteins?
   6 key words and vocabularies
     heterogeneity, three-dimensional structure,
     nuclear magnetic resonance (NMR), driving force,
     light-scattering instrument, fluorescence spectra,
     attractiveness, retentiveness, dehydration,
     microcalorimetry, boundary condition