RESEARCH PAPER
Influence of Acylation on the Adsorption of Insulin to Hydrophobic Surfaces
Charlotte Pinholt &Susanne Hostrup &Jens Thostrup Bukrinsky &Sven Frokjaer &Lene Jorgensen
Received:29August 2010/Accepted:8December 2010/Published online:29December 2010#Springer Science+Business Media,LLC 2010
ABSTRACT
Purpose T o study the effect of acylation on the adsorption of insulin to hydrophobic polystyrene beads.
Methods Adsorption isotherms for adsorption of insulin and acylated insulin to hydrophobic polystyrene beads were established,and the adsorption of the two proteins was compared further with isothermal titration calorimetry.In addition,the secondary structure and the association behavior of the two proteins were studied with circular dichroism.Results Insulin and acylated insulin adsorbed with high affinity to the hydrophobic polystyrene beads.More acylated insulin molecules than insulin molecules adsorbed per unit surface area from solutions containing monomer-dimer mixtures of acylated insulin and insulin,respectively.In contrast,no difference was observed in the number of insulin and acylated insulin molecules adsorbing per unit surface area,when
adsorption occurred from solutions containing monomer-dimer-hexamer mixtures.
Conclusion The influence of acylation on the adsorption behavior of insulin depends on the association degree of insulin,possibly due to a greater difference in hydrophobicity between monomeric insulin and acylated insulin than between the hexameric forms of these two proteins.
KEY WORDS acylated insulin .insulin .protein adsorption .ITC .CD
INTRODUCTION
Acylation is the covalent attachment of a fatty acid chain to proteins or peptides (hereinafter referred to as proteins).It is a naturally occurring post-translational modification,but in the last three decades the use of protein-engineered acylation to facilitate the delivery of protein drugs by increasing their plasma half life has gained increased interest.The prolonged plasma half is obtained by binding of the fatty acid chain to serum albumin in plasma and through sustained release from the local administration site (1–3).
Acylated protein drugs are,like other protein drugs,exposed to a diversity of solid-liquid interfaces during purification,production,storage and use.Typical hydro-phobic solid surfaces that protein solutions come into contact with during storage and use are closures,plungers,infusion bags,tubing and valves.Adsorption at interfaces during storage and use will result in protein loss from solution and may therefore lead to incomplete delivery to patients.Furthermore,adsorption may cause surface-induced structural changes in adsorbed and possibly desorbed protein molecules.Such structural changes may lead to aggregation and reduced biological activity and
Electronic Supplementary Material The online version of this article (doi:10.1007/s11095-010-0349-6)contains supplementary material,which is available to authorized users.
C.Pinholt :S.Frokjaer :L.Jorgensen
Department of Pharmaceutics and Analytical Chemistry
Faculty of Pharmaceutical Sciences,University of Copenhagen DK-2100Copenhagen,Denmark
S.Hostrup
CMC Diabetes Analysis and Formulation,Novo Nordisk A/S DK-2880Bagsvaerd,Denmark J.T .Bukrinsky
Novozymes Biopharma DK A/S DK-2880Bagsvaerd,Denmark C.Pinholt (*)
Faculty of Pharmaceutical Sciences Universitetsparken 2
DK-2100Copenhagen,Denmark e-mail:cp@farma.ku.dk
Pharm Res (2011)28:1031–1040DOI 10.1007/s11095-010-0349-6
potentially cause adverse immunological responses to the protein drug.Acylation will affect the protein characteristics and is therefore likely to affect the protein adsorption behavior at solid-liquid interfaces.Therefore,a thorough understanding of the affect of acylation on protein adsorption would be of great value in the future development of acylated protein drug products.
Insulin is a5808Da protein consisting of an A-chain and a B-chain linked by two disulfide bonds,with an additional intra-chain disulfide bond in the A-chain(4).In solution, insulin exhibits a complicated self-association behavior, where monomers exist in equilibrium with dimers and hexamers.The self-association of insulin is affected by a range of factors,including the insulin concentration,pH, ionic strength,divalent metal ions and various ligands(5).At concentrations below0.1μM,insulin exists as a monomer, whereas dimers are formed at higher concentrations(6).The hexamer form is promoted in the presence of Zn2+ions.In the presence of Zn2+ions and in the pH range4–8,hexamers are formed at concentrations above0.01mM(6),but in the absence of Zn2+ions,the hexamer is formed at concentrations above2mM(7).The monomer has hydrophobic amino acids exposed to the solution;therefore,it is thermodynamically favorable for two monomers to assemble into a dimer,where some of the hydrophobic amino acid residues are buried between the two insulin molecules(8).The formation of a heaxmer results in further burial of hydrophobic amino acid residues and the surface of a hexamer is almost entirely polar (8).When two monomers assemble,a shared anti-parallel β-sheet structure is formed(6),which makes the monomer-monomer interaction stronger than the interactions involved in the formation of hexamers(6,8).
Adsorption of insulin at solid-liquid interfaces has been studied with regard to varying hydrophobicity of the solid surface,association state of insulin,ionic strength and pH(9–11).Studies by Nilsson et al.(11) indicate that it is the monomeric form of insulin that adsorbs to a hydrophobic surface,while the adsorption plateau values observed by Mollmann et al.(9,10)are slightly higher than expected for a monolayer consisting of insulin monomers and correspond to monolayers consisting of a mixture of insulin monomers and dimers (9).To the best of our knowledge,the effect of acylation on the adsorption behavior of insulin has not been studied.
Today,an acylated insulin variant and an acylated GLP-1 variant are marketed,while acylation has been reported to increase the plasma half-life of leu-enkephalin(12), desmopressin(13,14),octreotide(15),salmon calciotonin (16)and interferon-alpha(17).
Lys B29(N-tetradecanoyl)des-(B30)human insulin,which is marketed under the name Levemir?,is a long-acting acylated human insulin variant in which Thr B30has been removed and a14-carbon myristoyl fatty acid has been covalently bound to the side chain of Lys B29(Fig.1).This 5917Da acylated insulin variant has a higher free energy of unfolding than insulin(18).The crystal structure of Lys B29 (N-tetradecanoyl)des-(B30)human insulin shows that the hydrophobic fatty acid group at the end of the chain has no significant effect on the conformation of the insulin monomer or its ability to form hexamers,but the fatty acid chains are involved in hydrophobic interaction between two adjacent hexamers(18).
The aim of this work was to examine how acylation of insulin influences the adsorption of insulin to hydrophobic polystyrene beads,using human insulin and Lys B29 (N-tetradecanoyl)des-(B30)human insulin as model proteins.Throughout the study,two different buffer systems were used,namely10mM NaH2PO4+1mM EDTA pH7.4,in which insulin and acylated insulin exist as a monomeric-dimeric equilibrium,and10mM NaH2PO4pH7.4,in which the two proteins exist as a monomeric-dimeric-hexameric equilibrium.Adsorption isotherms for the adsorption of the two proteins to polystyrene beads were compared,and isothermal titra-tion calorimetry was used to further compare their adsorption.Near-UV and far-UV circular dichroism were used to compare the secondary structure and association behavior of the two proteins.
MATERIALS AND METHODS
Materials
Freeze-dried bulk preparations of recombinant human insulin (insulin)and Lys B29-dodecanoyl des-(B30)insulin(acylated insulin)were kindly donated by Novo Nordisk A/S, Denmark.Both insulin and acylated insulin contained2.3 Zn2+ions per hexamer.In addition,the acylated insulin bulk contained one phenol molecule per protein molecule.Phenol can bind in the hydrophobic cavities between insulin dimers and thereby shift the hexamer configuration from the T-state to the more stable R-state(19),and this is also the case for acylated insulin(18).However,Zn needs to be present for insulin to form hexamers at the concentrations used in our experiments.Thus,the differences observed between insulin and acylated insulin at monomeric/dimeric conditions are unlikely to be a result of the presence of phenol in the acylated insulin solution.The T to R conformation change is accompanied by a large change in the250–260nm range of the near-UV spectrum(20).The near-UV signals measured for insulin and acylated insulin were all between1.3and1.6. These values are of the same range as the values reported by Olsen and Kaarsholm(18)when no phenol is present. Moreover,they show that when only one phenol molecule per acylated insulin molecule is present,the fraction of
1032Pinholt et al.
acylated insulin in the R-state is small (app.2%).Therefore,the phenol present in our acylated insulin samples is unlikely to affect the adsorption of acylated insulin.The isoelectric point of the insulin monomer has been determined to 5.4(21,22)and is expected to be even lower for the acylated insulin due to blocking of the positive charge at Lys B30.Therefore,insulin and the acylated insulin will both be negatively charged at pH 7.4.All water used was Milli-Q grade,and all other chemicals were of analytical grade and were obtained from commercial sources.Sample Preparation
Insulin was dispersed in water,and 0.1N HCl was added to dissolve the insulin,after which buffer was added and pH adjusted to 7.4with 0.1N NaOH.Acylated insulin was dissolved in water,buffer was added and pH was adjusted to 7.4with 0.1N HCl.The concentrations of the samples were determined from the absorbance at 276nm,using an extinction coefficient of 6200M ?1cm ?1(23).The buffer was either 10mM NaH 2PO 4pH 7.4or 10mM NaH 2PO 4+1mM EDTA pH 7.4.EDTA was added to bind Zn 2+ions and thereby prevent hexamer formation of insulin and acylated insulin,which can form in the presence of free Zn 2+ions.Addition of 1mM EDTA to a 300μM insulin solution will result in a free Zn 2+concentration of 8.67×10?16.(The concentration of free Zn 2+ions was calculated using MaxChelator (https://www.doczj.com/doc/cc5293828.html, ).)Polystyrene Beads
An 8w/v %suspension of polystyrene beads with sulphate functional groups on the surface suspended in distilled de-ionized water was obtained from Interfacial Dynamics Corporation (Eugene,OR,USA).Due to the sulphate groups,the surfaces of the polystyrene beads were partly negatively charged.The ionic strengths of the NaH 2PO 4and NaH 2PO 4+EDTA buffer solutions used for preparation
of polystyrene beads solutions were adjusted to make sure that the final suspensions of polystyrene beads had were in 10mM NaH 2PO 4pH 7.4or 10mM NaH 2PO 4+1mM EDTA pH 7.4.
Circular Dichroism (CD)
Far-UV and near-UV CD spectra were obtained on a Jasco-J-810circular dichroism spectrophotometer (Jasco,Tokyo,Japan).One-hundred-μM insulin and acylated insulin samples were scanned in a 0.05mm cell from 250to 180nm,and 300μM samples were scanned in a 5mm cell from 350to 250nm to obtain far-UV and near-UV spectra,respectively.A bandwidth of 1nm,a response time of 4s,a data pitch of 0.2nm and a scanning speed of 50nm/min were used.Each spectrum is an accumulation of 10scans.Spectra of the appropriate reference solutions were recorded and subtracted from each protein spectrum.The molar CD signal Δεwas expressed in M ?1cm ?1with far-UV and near-UV data normalized to the molar concentration of protein and peptide bond,respectively.Adsorption Isotherms
Adsorption isotherms were established for insulin and acylated insulin adsorption to polystyrene beads.For each protein,solutions of varying concentration were added to 3.2ml Beckman centrifuge polycarbonate tubes containing beads with at total surface area of 0.1m 2.A total volume of 3ml was added to each tube.At high concentration and in the presence of Zn 2+ions,insulin and acylated insulin can form hexamers and di-hexamers,respectively.A 10mM NaH 2PO 4buffer with pH 7.4containing 1mM EDTA was used in order to bind the Zn 2+ions and thereby prevent formation of hexamers.The samples were left to adsorb at room temperature for 1h,after which they were centrifuged with 100,000rpm (550,000g)for 30min at 25°C in a Beckman Coulter Optima Max-E Ultra Centrifuge using
a
Fig.1Primary structure of acylated human insulin variant.Thr B30has been removed,and a 14-carbon myristoyl fatty acid has been covalently bound to the side chain of Lys B29.Modified from (3).
Influence of Acylation on Adsorption of Insulin 1033
TLA110rotor(Beckman Coulter,Fullerton,CA,USA). Subsequently,the supernatant was removed and the protein concentration determined by UV spectroscopy usingε276= 6200M?1cm?1(1,23),and the adsorbed amount was then determined by mass balance.In order to study whether desorption of insulin and acylated insulin occurred,the beads were re-suspended in3ml buffer and left overnight.The next day,the samples were centrifuged,and the concentra-tion in the supernatant was determined.
Isothermal Titration Calorimetry Experiments Isothermal titration calorimetry measurements were per-formed with a Nano ITC2G from TA Instruments(Newcastle, DE,USA).In a typical experiment,the sample cell was filled with a suspension of polystyrene beads yielding a surface area of0.11m2.The suspension of polystyrene beads was titrated with5μL aliquots of300μM insulin or acylated insulin.The temperature was25°C,and the reference cell was filled with water.All solutions were degassed by stirring under vacuum before use.Four different experiments were performed: titration of insulin and acylated insulin into beads in 10mM NaH2PO4+1mM EDTA pH7.4and titration of insulin and acylated insulin into beads in10mM NaH2PO4 pH7.4.The titration of insulin into beads suspended in 10mM NaH2PO4+1mM EDTA pH7.4was performed three times to examine the reproducibility.Since the reproducibility was high,the other experiments were per-formed only once or twice.Heat of dilution was subtracted for all heat signals before they were integrated using the NanoAnalyze software from TA instruments.
RESULTS
Circular Dichroism
The far-UV spectra for insulin and acylated insulin in 10mM NaH2PO4+1mM EDTA pH7.4showed structures dominated byα-helix with a strong negative CD signals at208and222nm and a strong positive CD signal around195nm(Fig.2a).The negative CD signal at 208nm and222nm as well as the positive CD signal around195nm were smaller for acylated insulin than for insulin,while the positive CD signal from190–180nm was higher for acylated insulin than for insulin.Furthermore, the crossover point at the x-axis and the positive band at 195nm were blue-shifted for acylated insulin.In10mM NaH2PO4pH7.4(Fig.2b),far-UV spectra similar to those in10mM NaH2PO4+1mM EDTA pH7.4were https://www.doczj.com/doc/cc5293828.html,pared to insulin,the spectra for acylated insulin had a slightly larger negative CD signal at208nm and a slightly smaller negative CD signal at222nm.Furthermore,the crossover point at the x-axis and the positive band at195nm were blue-shifted for acylated insulin compared to insulin.Although differences in the far-UV CD spectra for insulin and acylated insulin were observed,the spectra for both proteins were dominated by α-helical structure in both10mM NaH2PO4+1mM EDTA pH7.4and10mM NaH2PO4pH7.4.
Near-UV CD spectra of insulin and acylated insulin, respectively,in both buffers are shown in Fig.3.All four spectra are dominated by a tyrosyl signal with a minimum at276nm.Insulin dimers and hexamers exhibit a characteristic tyrosyl CD signal at approximately276nm. The two main contributors to this signal are interactions in the monomer-monomer interface of the dimers and in the dimer-dimer interfaces of the hexamers(24,25).The near-UV CD spectra for both insulin variants showed a larger negative CD signal around276nm in10mM NaH2PO
4 Fig.2Far-UV CD spectra of insulin(black line)and acylated insulin(gray dashed line)obtained on100μM solutions in0.5mm cells.(A)In10mM NaH2PO4+1mM EDTA pH7.4.(B)In10mM NaH2PO4pH7.4.
1034Pinholt et al.
pH 7.4than in 10mM NaH 2PO 4+1mM EDTA pH 7.4,indicating that the monomer-dimer-hexamer equilibrium was shifted further toward the hexamer form when the solution contained Zn 2+ions.The observed difference in the intensity of the CD signal for insulin and acylated insulin is in accordance with previous near-UV CD measurements on Zn-free human insulin and Zn-free acylated insulin (18).Adsorption Isotherms
Adsorption and desorption isotherms were obtained for adsorption of insulin and acylated insulin to polystyrene beads in 10mM NaH 2PO 4+1mM EDTA,pH 7.4(Fig.4).The initial part of the adsorption isotherms merges with the y-axis,indicating that both proteins adsorb with high affinity
to the polystyrene beads.For insulin and acylated insulin,adsorption saturation was reached above approximately 12μM with plateau values of 2.4±0.3×10?4mol/m 2and 3.2±0.110?4mol/m 2,respectively.For insulin,the desorp-tion isotherm could generally not be distinguished from the adsorption isotherm.For two points on the isotherm the average values for the adsorption and desorption differed,but the observed differences were within the standard deviation and therefore no indication of desorption.Slight desorption was observed for acylated insulin yielding a plateau value of 3.0±0.210?4mol/m 2for the desorption isotherm.Even though a slight desorption was observed for acylated insulin,the plateau value after desorption was still higher for acylated insulin than for insulin.Isothermal Titration Calorimetry
Representative raw data for titration of insulin and acylated insulin into 10mM NaH 2PO 4+1mM EDTA pH 7.4and into a suspension of polystyrene beads in the same buffer are shown in Fig.5.Blank titrations of insulin into buffer revealed small exothermic heat flows that gradually de-creased with the number of injections,and towards the end a small endothermic heat flow was observed after each injection.The size of the heat flow signals for the blank titration of insulin into buffer indicates that the contribution from dissociation of insulin dimers will have minute influence on the experiments with insulin.For titration of acylated insulin into buffer,a steady small endothermic heat flow was observed for each injection,indicating that heat flow from dissociation for acylated insulin will not affect the results for titrations of acylated insulin into a suspension of
polystyrene
Fig.3Near-UV CD spectra of insulin and acylated insulin obtained on 300μM solutions in 0.5mm cells.(A )Insulin in 10mM NaH 2PO 4+1mM EDTA pH 7.4(black line )and in 10mM NaH 2PO 4pH 7.4(black dashed line ).(B )Acylated insulin in 10mM NaH 2PO 4+1mM EDTA pH 7.4(gray line )and in 10mM NaH 2PO 4pH 7.4(gray dashed line
).
Fig.4Adsorption isotherms for absorption of insulin ()and acylated insulin ()to polystyrene particles in 10mM NaH 2PO 4+1mM EDTA pH 7.4.Insulin ()and acylated insulin (after desorption.If no desorption was detected,the adsorbed amounts per unit surface area is shown only with an open square.
Influence of Acylation on Adsorption of Insulin 1035
beads.Titration of insulin and acylated insulin into the suspension of polystyrene beads was accompanied by exothermic heat flows,which after a few injections decreased gradually until a small endothermic heat flow was observed following an injection.A steady heat flow was obtained after 13injections for insulin,whereas it was obtained after 16injections for acylated insulin.Both the enthalpogram for insulin and acylated insulin were s-shaped (Fig.6).
In 10mM NaH 2PO 4pH 7.4,s-shaped enthalpograms were also observed for insulin and acylated insulin titrations into polystyrene beads (Fig.7).The enthalpogram for insulin was similar to the enthalpogram obtained in 10mM NaH 2PO 4+1mM EDTA pH 7.4.The enthalpogram for titration of acylated insulin into the suspension of polystyrene beads in 10mM NaH 2PO 4+pH 7.4was accompanied by a lower exothermic heat flow than upon titration into the suspension of polystyrene beads in 10mM NaH 2PO 4+1mM EDTA pH 7.4.The heat flow signals for blank
titration of insulin and acylated insulin,respectively,into 10mM NaH 2PO 4pH 7.4were similar to the heat flows observed for titrations into 10mM NaH 2PO 4+1mM EDTA (data not shown).
DISCUSSION
Due to the self-association behavior of insulin and acylated insulin,their adsorption behaviors were compared in two buffer systems.First,in a 10mM NaH 2PO 4+1mM EDTA pH 7.4buffer,EDTA binds the Zn 2+ions and thereby prevents hexamer formation,resulting in a solution con-taining only monomers and https://www.doczj.com/doc/cc5293828.html,ing the dimer association constant 7.5×105M ?1(26)for human insulin at pH 7.0,the monomer-dimer distribution at 300μM and 8μM is calculated to 10:90and 50:50,respectively.Below 1μM,the solution will be dominated by monomers.
Near-
Fig.5Raw heat signals from isothermal titration calorimetric experiments obtained at 25°C.T op:titration of 5μL aliquots of 300μM insulin ()and 300μM acylated insulin ()into 10mM NaH 2PO 4+1mM EDTA,pH 7.4.Bottom:titration of 5μL aliquots of 300μM insulin ()and 300μM acylated insulin ()into a suspension of polystyrene beads with a total surface area of 0.11m 2in the cell.
1036Pinholt et al.
UV CD and NMR on insulin and acylated insulin has shown that the acyl side-chain interferes with protein-protein recognition in the dimer interface,thereby increas-ing the dimer dissociation constant of acylated insulin of one order of magnitude or more compared to insulin (18).In the second buffer,10mM NaH 2PO 4pH 7.4,both proteins will be present as monomer-dimer-hexamer equilibriums.In a solution of bovine insulin containing 2Zn 2+ions per insulin hexamer and at pH 7.0,more than 75%of the total insulin has been shown to be hexameric at a concentration above 17μM (27).
Adsorption from Solutions Containing Monomers and Dimers
Driving Forces and Reversibility
After attachment to a surface,protein molecules may form numerous contacts with a sorbent surface (28),likely leading to high-affinity adsorption.Both insulin and acylated insulin reached adsorption saturation at concentrations above approximately 12μM (Fig.4)under conditions of electro-static repulsion.This indicates that both proteins have a high affinity for the polystyrene beads and that hydrophobic interactions are the dominating driving force for adsorption,which is in accordance with previous findings for adsorption of insulin to hydrophobic Teflon beads,a hydrophobic PTFE-like surface and a hydrophobic methylated silica surface (9–11).This observation is further supported by the slight negative slope observed for ΔH versus temperature (ΔC p ),which points to the hydrophobic effect as a driving force for the absorption (data not shown)(29).
For most proteins adsorbing to a surface with high affinity,a portion of the protein molecules adsorb irreversibly toward dilution (30),while another fraction of the protein molecules are only loosely adsorbed above monolayer coverage.Such loosely adsorbed protein molecules are expected to be only partly in contact with the surface or other adsorbed protein molecules,and they may be removed by dilution with buffer (31,32).The slight desorption of acylated insulin molecules revealed by the desorption isotherm indicates that only loosely adsorbed acylated insulin molecules were removed by dilution with 10mM NaH 2PO 4+1mM EDTA pH 7.4,thereby indicating that acylated insulin adsorbs irreversibly to the hydrophobic polystyrene particles.No desorption of insulin could be measured upon dilution with 10mM NaH 2PO 4+1mM EDTA pH 7.4,showing that insulin adsorbs irreversibly to the hydrophobic polystyrene particles.This is in accordance with previous findings for adsorption of insulin to hydrophobic Teflon beads and hydrophobic PTFE-like surfaces (9,10).It is possible that more acylated insulin molecules than insulin molecules adsorb loosely,due to interactions between the fatty acid chains of adjacent molecules.Adsorbed Amounts
The plateau values for the adsorption isotherms after desorption were 2.4±0.3×10?4mol/m 2(1.4mg/m 2)and 3.0±0.210?4mol/m 2(1.8mg/m 2)for insulin and acylated insulin,respectively (Fig 4).The amount of insulin molecules adsorbing per unit surface area to different hydrophobic surfaces has been reported to be between 0.8and 1.7mg/m 2(9–11,31).It has been proposed that at higher protein concentration in solution,a surface
becomes
Fig.7Isothermal titration calorimetric data for the titration of 300μM insulin ()and 300μM acylated insulin ()into a suspension of polystyrene beads in 10mM NaH 2PO 4,pH 7.4at 25°C.The raw heat signal peaks have been integrated to obtain the enthalpy change per mole of protein injected.The total surface area of the polystyrene beads in the cell was 0.11m 2
.
Fig.6Isothermal titration calorimetric data for the titration of 300μM insulin ()and 300μM acylated insulin ()into a suspension of polystyrene beads in 10mM NaH 2PO 4+1mM EDTA,pH 7.4at 25°C.The raw heat signal peaks have been integrated to obtain the enthalpy change per mole of protein injected.The total surface area of the polystyrene beads in the cell was 0.11m 2.
Influence of Acylation on Adsorption of Insulin 1037
covered more quickly,allowing less time for the molecules to relax at the surface(33).This may affect the orientation and the conformation of the adsorbed protein molecules and thereby the amount of molecules adsorbing per unit surface area(34).Therefore,the estimated amount of protein molecules adsorbing per unit surface area tends to depend on the experimental setup.In addition,the adsorption plateau value of a protein will depend on the hydrophobicity of the surface(35).Therefore,the adsorp-tion plateau value for insulin of1.4mg/m2,obtained from our adsorption isotherm(Fig.4),corresponds well with the previous reported values of0.8–1.7mg/m2.
ITC data have by several authors been fitted to reversible isotherm equations.However,other authors have pointed out that the fitting of experimental data on equilibrium amounts of adsorbed protein to reversible isotherm equations is questionable for an irreversible adsorbed amount of protein(34,36).As it is evident from our adsorption isotherms that the adsorption of both insulin and acylated insulin is irreversible,the ITC data have not been fitted.From the ITC experiments,it was observed that16injections of acylated insulin were needed to reach saturation in the ITC cell,whereas only13injections of insulin were needed(Fig.6).This indicates that acylation increases the number of insulin molecules adsorbing per unit surface area and is in accordance with the findings from the adsorption isotherms.Several parameters can influence the number of protein molecules adsorbing per unit surface area,including the size,charge and hydrophobicity of the protein.The molar mass of acylated insulin is5917Da compared to the molar mass of5808Da for insulin.Assuming that the higher molar mass of acylated insulin is synonymous with a bigger size, acylation of insulin would be expected to decrease the number of insulin molecules adsorbing per unit surfaces area.A structurally stable protein is less prone to undergo structural changes upon adsorption than a more structurally labile protein and is therefore likely to cover a smaller surface area(37).Acylation has been shown to increase the Gibbs free energy of unfolding for acylated insulin(18)and may therefore lead to a closer packing of acylated insulin.It is also possible that it is the fatty acid chain of acylated insulin that interacts with the hydropho-bic polystyrene beads and that an acylated insulin molecule adsorbed in this manner will take up less surface area than an insulin molecule.
Acylated insulin is likely to have a higher net negative charge than insulin,because the fatty acid chain is attached to the Lys B30side chain,which is positively charged in insulin at pH7.4.The higher negative charge will decrease the affinity of acylated insulin towards the hydrophobic and partly negatively charged polystyrene beads.However,acylation increases the hydrophobicity of insulin and is therefore likely to increase the affinity of insulin for the hydrophobic polystyrene beads.Since the major driving force for the adsorption is hydrophobic interactions,the contribution from the increased hydro-phobicity should dominate over the increased electrostatic repulsion due to difference in charge.
Adsorption from Solutions Containing Monomers, Dimers and Hexamers
The enthalpogram obtained for titration of insulin into a suspension of hydrophobic polystyrene beads in10mM NaH2PO4+1mM EDTA pH7.4was similar to the enthalpogram obtained for titration of insulin into a suspension of hydrophobic polystyrene particles in10mM NaH2PO4pH7.4(Figs.6and7).This indicates that the presence of2Zn2+ions per hexamer did not affect the number of insulin molecules adsorbing per unit surface area,which is in accordance with a previous study where the adsorbed amounts of Zn-free human insulin and human insulin containing2Zn2+ions per hexamer were compared by ellipsometry(11).In contrast,fever injections of acylated insulin were needed to obtain saturation in the ITC experiment in10mM NaH2PO4pH7.4as compared to the ITC experiment in10mM NaH2PO4+1mM EDTA pH7.4(Figs.6and7).This indicates that the adsorption of acylated insulin to hydrophobic beads is influenced by the presence of free Zn2+ions and thereby influenced by the association behavior of acylated insulin.For the ITC experiments in10mM NaH2PO4pH7.4,both insulin and acylated insulin reached saturation after13injections, indicating that the same amount of insulin and acylated insulin molecules adsorb to the hydrophobic polystyrene beads per unit surface area.The same amount of insulin and acylated insulin molecules has also been found to adsorb to hydrophobic gold surface in10mM NaH2PO4 pH7.4(see Supplementary Material I).
Due to the hydrophobic character of the fatty acid chain, acylation will increase the hydrophobicity of insulin.It is mainly the nonpolar residues of the insulin monomer that are involved in the association into dimers and hexamers, and the surface of the hexamer is almost entirely polar(8). In solutions containing acylated insulin hexamers,two acylated insulin hexamers can form di-hexamer,in which three fatty acid chains from each of two adjacent hexamers interact(38)and thereby shield the fatty acid chains from the water.Therefore,the differences in hydrophobicity of insulin and acylated insulin will be smaller in a solution containing monomers,dimers and hexamers than in a solution containing only monomers and dimers.We suggest that the decreased amount of acylated insulin molecules adsorbing per unit surface area in the presence of Zn2+ions is a result of the decreased hydrophobicity of the acylated
1038Pinholt et al.
insulin due to the hexamer and di-hexamer formation.It cannot be ruled out that the decreased hydrophobicity of insulin due to hexamer formation also affects the adsorbed amount of insulin.However,this could not be measured with the ITC setup used.
CONCLUSION
Insulin and acylated insulin were observed to adsorb with high affinity to hydrophobic polystyrene beads,and hydrophobic interactions seemed to be the major driving forces for the adsorption.The effect of acylation on the amount of insulin molecules adsorbing per unit surface area to hydrophobic polystyrene beads was dependent on the association degree of insulin.Upon adsorption from solutions containing monomeric and dimeric insulin and acylated insulin,respectively,acylation increased the num-ber of insulin molecules adsorbing per unit surface area.In contrast,no differences in the adsorbed amounts of insulin and acylated insulin per unit surface area were detected upon adsorption from solutions of the two proteins containing monomers,dimers and hexamers.We suggest that this difference in the effect of acylation on the amount of insulin molecules adsorbing per unit surface area is due to a greater difference in hydrophobicity between mono-meric insulin and acylated insulin than between the hexameric forms of these two proteins. ACKNOWLEDGMENT
Financial support from the Drug Research Academy is gratefully acknowledged.In addition,the Advanced Tech-nology Foundation is acknowledged for financing the ITC. We are thankful to Novo Nordisk A/S for providing all the protein material used in this study.Dennis Isaksen is greatly acknowledged for performing the adsorption isotherm experiments and for assistance with the ITC experiments. REFERENCES
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一般过去式 时间状语：yesterday just now (刚刚) the day before three days ag0 a week ago in 1880 last month last year 1. I was in the classroom yesterday. I was not in the classroom yesterday. Were you in the classroom yesterday. 2. They went to see the film the day before. Did they go to see the film the day before. They did go to see the film the day before. 3. The man beat his wife yesterday. The man didn’t beat his wife yesterday. 4. I was a high student three years ago. 5. She became a teacher in 2009. 6. They began to study english a week ago 7. My mother brought a book from Canada last year. 8.My parents build a house to me four years ago . 9.He was husband ago. She was a cooker last mouth. My father was in the Xinjiang half a year ago. 10.My grandfather was a famer six years ago. 11.He burned in 1991
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高中英语~词性~句子成分~语法构成 第一章节：英语句子中的词性 1.名词：n. 名词是指事物的名称，在句子中主要作主语.宾语.表语.同位语。 2.形容词;adj. 形容词是指对名词进行修饰~限定~描述~的成份，主要作定语.表语.。形容词在汉语中是（的）.其标志是: ous. Al .ful .ive。. 3.动词：vt. 动词是指主语发出的一个动作，一般用来作谓语。 4.副词：adv. 副词是指表示动作发生的地点. 时间. 条件. 方式. 原因. 目的. 结果.伴随让步. 一般用来修饰动词. 形容词。副词在汉语中是（地）.其标志是：ly。 5.代词：pron. 代词是指用来代替名词的词，名词所能担任的作用，代词也同样.代词主要用来作主语. 宾语. 表语. 同位语。 6.介词：prep.介词是指表示动词和名次关系的词，例如：in on at of about with for to。其特征：
介词后的动词要用—ing形式。介词加代词时，代词要用宾格。例如：give up her（him）这种形式是正确的，而give up she（he）这种形式是错误的。 7.冠词：冠词是指修饰名词，表名词泛指或特指。冠词有a an the 。 8.叹词：叹词表示一种语气。例如：OH. Ya 等 9.连词：连词是指连接两个并列的成分，这两个并列的成分可以是两个词也可以是两个句子。例如：and but or so 。 10.数词：数词是指表示数量关系词，一般分为基数词和序数词 第二章节：英语句子成分 主语：动作的发出者，一般放在动词前或句首。由名词. 代词. 数词. 不定时. 动名词. 或从句充当。 谓语：指主语发出来的动作，只能由动词充当，一般紧跟在主语后面。 宾语：指动作的承受着，一般由代词. 名词. 数词. 不定时. 动名词. 或从句充当. 介词后面的成分也叫介词宾语。 定语：只对名词起限定修饰的成分，一般由形容
M A: Has the case been closed yet? B: No, the magistrate still needs to decide the outcome. magistrate n.地方行政官，地方法官，治安官 A: I am unable to read the small print in the book. B: It seems you need to magnify it. magnify vt.１．放大，扩大；２．夸大，夸张 A: That was a terrible storm. B: Indeed, but it is too early to determine the magnitude of the damage. magnitude n.１．重要性，重大；２．巨大，广大 A: A young fair maiden like you shouldn’t be single. B: That is because I am a young fair independent maiden. maiden n.少女，年轻姑娘，未婚女子 a.首次的，初次的 A: You look majestic sitting on that high chair. B: Yes, I am pretending to be the king! majestic a.雄伟的，壮丽的，庄严的，高贵的 A: Please cook me dinner now. B: Yes, your majesty, I’m at your service. majesty n.１．［Ｍ－］陛下（对帝王，王后的尊称）；２．雄伟，壮丽，庄严 A: Doctor, I traveled to Africa and I think I caught malaria. B: Did you take any medicine as a precaution? malaria n.疟疾 A: I hate you! B: Why are you so full of malice? malice n.恶意，怨恨 A: I’m afraid that the test results have come back and your lump is malignant. B: That means it’s serious, doesn’t it, doctor? malignant a.１．恶性的，致命的；２．恶意的，恶毒的 A: I’m going shopping in the mall this afternoon, want to join me? B: No, thanks, I have plans already. mall n.（由许多商店组成的）购物中心 A: That child looks very unhealthy. B: Yes, he does not have enough to eat. He is suffering from malnutrition.
意见应以事实为根据. 3 来自辞典例句 192. The bombers swooped ( down ) onthe air base. 轰炸机 突袭 空军基地. 来自辞典例句 193. He mounted their engines on a rubber base. 他把他们的发动机装在一个橡胶垫座上. 14 来自辞典例句 194. The column stands on a narrow base. 柱子竖立在狭窄的地基上. 14 来自辞典例句 195. When one stretched it, it looked like grey flakes on the carvas base. 你要是把它摊直, 看上去就象好一些灰色的粉片落在帆布底子上. 18 来自辞典例句 196. Economic growth and human well - being depend on the natural resource base that supports all living systems. 经济增长和人类的福利依赖于支持所有生命系统的自然资源. 12 1 来自辞典例句 197. The base was just a smudge onthe untouched hundred - mile coast of Manila Bay. 那基地只是马尼拉湾一百英里长安然无恙的海岸线上一个硝烟滚滚的污点. 6 来自辞典例句 198. You can't base an operation on the presumption that miracles are going to happen. 你不能把行动计划建筑在可能出现奇迹的假想基础上.
英语造句大全English sentence 在句子中，更好的记忆单词！ 1、(1)、able adj. 能 句子：We are able to live under the sea in the future. (2)、ability n. 能力 句子：Most school care for children of different abilities. (3)、enable v. 使。。。能句子：This pass enables me to travel half-price on trains. 2、(1)、accurate adj. 精确的句子：We must have the accurate calculation. (2)、accurately adv. 精确地 句子：His calculation is accurately. 3、(1)、act v. 扮演 句子：He act the interesting character. （2）、actor n. 演员 句子：He was a famous actor. (3)、actress n. 女演员 句子：She was a famous actress. (4)、active adj. 积极的 句子：He is an active boy. 4、add v. 加 句子：He adds a little sugar in the milk. 5、advantage n. 优势 句子：His advantage is fight. 6、age 年龄n. 句子：His age is 15. 7、amusing 娱人的adj. 句子：This story is amusing. 8、angry 生气的adj. 句子：He is angry. 9、America 美国n.
主语+谓语 1. 理解主谓结构 1) The students arrived. The students arrived at the park. 2) They are listening. They are listening to the music. 3) The disaster happened. 2．体会状语的位置 1) Tom always works hard. 2) Sometimes I go to the park at weekends.. 3) The girl cries very often. 4) We seldom come here. The disaster happened to the poor family. 3. 多个状语的排列次序 1) He works. 2) He works hard. 3) He always works hard. 4) He always works hard in the company. 5) He always works hard in the company recently. 6) He always works hard in the company recently because he wants to get promoted. 4. 写作常用不及物动词 1. ache My head aches. I’m aching all over. 2. agree agree with sb. about sth. agree to do sth. 3. apologize to sb. for sth. 4. appear (at the meeting, on the screen) 5. arrive at / in 6. belong to 7. chat with sb. about sth. 8. come (to …) 9. cry 10. dance 11. depend on /upon 12. die 13. fall 14. go to … 15. graduate from 16. … happen 17. laugh 18. listen to... 19. live 20. rise 21. sit 22. smile 23. swim 24. stay (at home / in a hotel) 25. work 26. wait for 汉译英： 1.昨天我去了电影院。 2.我能用英语跟外国人自由交谈。 3.晚上7点我们到达了机场。 4.暑假就要到了。 5.现在很多老人独自居住。 6.老师同意了。 7.刚才发生了一场车祸。 8.课上我们应该认真听讲。9. 我们的态度很重要。 10. 能否成功取决于你的态度。 11. 能取得多大进步取决于你付出多少努力。 12. 这个木桶能盛多少水取决于最短的一块板子的长度。
【it's time to和it's time for】 ——————这其实是一个句型,只不过后面要跟不同的东西. ——————It's time to跟的是不定式（to do）.也就是说,要跟一个动词,意思是“到做某事的时候了”.如： It's time to go home. It's time to tell him the truth. ——————It's time for 跟的是名词.也就是说,不能跟动词.如： It's time for lunch.(没必要说It's time to have lunch) It's time for class.(没必要说It's time to begin the class.) They can't wait to see you Please ask liming to study tonight. Please ask liming not to play computer games tonight. Don’t make/let me to smoke I can hear/see you dance at the stage You had better go to bed early. You had better not watch tv It’s better to go to bed early It’s best to run in the morning I am enjoy running with music. With 表伴随听音乐 I already finish studying You should keep working. You should keep on studying English Keep calm and carry on 保持冷静继续前行二战开始前英国皇家政府制造的海报名字 I have to go on studying I feel like I am flying I have to stop playing computer games and stop to go home now I forget/remember to finish my homework. I forget/remember cleaning the classroom We keep/percent/stop him from eating more chips I prefer orange to apple I prefer to walk rather than run I used to sing when I was young What’s wrong with you There have nothing to do with you I am so busy studying You are too young to na?ve I am so tired that I have to go to bed early
《The Kite Runner》追风筝的人--------------------------------美句摘抄 1.I can still see Hassan up on that tree, sunlight flickering through the leaves on his almost perfectly round face, a face like a Chinese doll chiseled from hardwood: his flat, broad nose and slanting, narrow eyes like bamboo leaves, eyes that looked, depending on the light, gold, green even sapphire 翻译：我依然能记得哈桑坐在树上的样子，阳光穿过叶子，照着他那浑圆的脸庞。他的脸很像木头刻成的中国娃娃，鼻子大而扁平，双眼眯斜如同竹叶，在不同光线下会显现出金色、绿色，甚至是宝石蓝。 E.g.: A shadow of disquiet flickering over his face. 2.Never told that the mirror, like shooting walnuts at the neighbor's dog, was always my idea. 翻译：从来不提镜子、用胡桃射狗其实都是我的鬼主意。E.g.:His secret died with him, for he never told anyone. 3.We would sit across from each other on a pair of high
一、翻译 1. The idea of consciously seeking out a special title was new to me., but not without appeal. 让我自己挑选自己最喜欢的书籍这个有意思的想法真的对我具有吸引力。 2.I was plunged into the aching tragedy of the Holocaust, the extraordinary clash of good, represented by the one decent man, and evil. 我陷入到大屠杀悲剧的痛苦之中，一个体面的人所代表的善与恶的猛烈冲击之中。 3.I was astonished by the the great power a novel could contain. I lacked the vocabulary to translate my feelings into words. 我被这部小说所包含的巨大能量感到震惊。我无法用语言来表达我的感情（心情）。 4，make sth. long to short长话短说 5.I learned that summer that reading was not the innocent(简单的) pastime(消遣) I have assumed it to be., not a breezy, instantly forgettable escape in the hammock(吊床)，( though I’ ve enjoyed many of those too ). I discovered that a book, if it arrives at the right moment, in the proper season, will change the course of all that follows. 那年夏天，我懂得了读书不是我认为的简单的娱乐消遣，也不只是躺在吊床上，一阵风吹过就忘记的消遣。我发现如果在适宜的时间、合适的季节读一本书的话，他将能改变一个人以后的人生道路。 二、词组造句 1. on purpose 特意，故意 This is especially true here, and it was ~. (这一点在这里尤其准确，并且他是故意的) 2.think up 虚构，编造，想出 She has thought up a good idea. 她想出了一个好的主意。 His story was thought up. 他的故事是编出来的。 3. in the meantime 与此同时 助记：in advance 事前in the meantime 与此同时in place 适当地... In the meantime, what can you do? 在这期间您能做什么呢？ In the meantime, we may not know how it works, but we know that it works. 在此期间，我们不知道它是如何工作的，但我们知道，它的确在发挥作用。 4.as though 好像，仿佛 It sounds as though you enjoyed Great wall. 这听起来好像你喜欢长城。 5. plunge into 使陷入 He plunged the room into darkness by switching off the light. 他把灯一关，房
The effective sentences:(improve the sentences!) 1.She hopes to spend this holiday either in Shanghai or in Suzhou. 2.Showing/to show sincerity and to keep/keeping promises are the basic requirements of a real friend. 3.I want to know the space of this house and when it was built. I want to know how big this house is and when it was built. I want to know the space of this house and the building time of the house. 4.In the past ten years,Mr.Smith has been a waiter,a tour guide,and taught English. In the past ten years,Mr.Smith has been a waiter,a tour guide,and an English teacher. 5.They are sweeping the floor wearing masks. They are sweeping the floor by wearing masks. wearing masks,They are sweeping the floor. 6.the drivers are told to drive carefully on the radio. the drivers are told on the radio to drive carefully 7.I almost spent two hours on this exercises. I spent almost two hours on this exercises. 8.Checking carefully,a serious mistake was found in the design. Checking carefully,I found a serious mistake in the design.
M1 U1 一. 把下列短语填入每个句子的空白处（注意所填短语的形式变化）： add up (to) be concerned about go through set down a series of on purpose in order to according to get along with fall in love (with) join in have got to hide away face to face 1 We’ve chatted online for some time but we have never met ___________. 2 It is nearly 11 o’clock yet he is not back. His mother ____________ him. 3 The Lius ___________ hard times before liberation. 4 ____________ get a good mark I worked very hard before the exam. 5 I think the window was broken ___________ by someone. 6 You should ___________ the language points on the blackboard. They are useful. 7 They met at Tom’s party and later on ____________ with each other. 8 You can find ____________ English reading materials in the school library. 9 I am easy to be with and _____________my classmates pretty well. 10 They __________ in a small village so that they might not be found. 11 Which of the following statements is not right ____________ the above passage? 12 It’s getting dark. I ___________ be off now. 13 More than 1,000 workers ___________ the general strike last week. 14 All her earnings _____________ about 3,000 yuan per month. 二.用以下短语造句： 1.go through 2. no longer/ not… any longer 3. on purpose 4. calm… down 5. happen to 6. set down 7. wonder if 三. 翻译： 1.曾经有段时间，我对学习丧失了兴趣。（there was a time when…） 2. 这是我第一次和她交流。（It is/was the first time that …注意时态） 3.他昨天公园里遇到的是他的一个老朋友。（强调句） 4. 他是在知道真相之后才意识到错怪女儿了。（强调句） M 1 U 2 一. 把下列短语填入每个句子的空白处（注意所填短语的形式变化）： play a …role (in) because of come up such as even if play a …part (in) 1 Dujiangyan(都江堰) is still ___________in irrigation(灌溉) today. 2 That question ___________ at yesterday’s meeting. 3 Karl Marx could speak a few foreign languages, _________Russian and English. 4 You must ask for leave first __________ you have something very important. 5 The media _________ major ________ in influencing people’s opinion s. 6 _________ years of hard work she looked like a woman in her fifties. 二.用以下短语造句： 1.make (good/full) use of 2. play a(n) important role in 3. even if 4. believe it or not 5. such as 6. because of
English sentence 1、(1)、able adj. 能 句子：We are able to live under the sea in the future. (2)、ability n. 能力 句子：Most school care for children of different abilities. (3)、enable v. 使。。。能 句子：This pass enables me to travel half-price on trains. 2、(1)、accurate adj. 精确的 句子：We must have the accurate calculation. (2)、accurately adv. 精确地 句子：His calculation is accurately. 3、(1)、act v. 扮演 句子：He act the interesting character.（2）、actor n. 演员 句子：He was a famous actor. (3)、actress n. 女演员 句子：She was a famous actress. (4)、active adj. 积极的 句子：He is an active boy. 4、add v. 加 句子：He adds a little sugar in the milk. 5、advantage n. 优势 句子：His advantage is fight. 6、age 年龄n. 句子：His age is 15. 7、amusing 娱人的adj. 句子：This story is amusing. 8、angry 生气的adj. 句子：He is angry. 9、America 美国n. 句子：He is in America. 10、appear 出现v. He appears in this place. 11. artist 艺术家n. He is an artist. 12. attract 吸引 He attracts the dog. 13. Australia 澳大利亚 He is in Australia. 14.base 基地 She is in the base now. 15.basket 篮子 His basket is nice. 16.beautiful 美丽的 She is very beautiful. 17.begin 开始 He begins writing. 18.black 黑色的 He is black. 19.bright 明亮的 His eyes are bright. 20.good 好的 He is good at basketball. 21.British 英国人 He is British. 22.building 建造物 The building is highest in this city 23.busy 忙的 He is busy now. 24.calculate 计算 He calculates this test well. 25.Canada 加拿大 He borns in Canada. 26.care 照顾 He cared she yesterday. 27.certain 无疑的 They are certain to succeed. 28.change 改变 He changes the system. 29.chemical 化学药品