溶液中和气-液界面处酶法交联β-乳球蛋白-结构限制
Enzymatic cross-linking of b -lactoglobulin in solution and at air e water interface:Structural constraints
Dilek Ercili-Cura a ,*,Riitta Partanen a ,Fiona Husband b ,Mike Ridout b ,Adam Macierzanka b ,Martina Lille a ,Harry Boer a ,Raija Lantto a ,Johanna Buchert a ,Alan R.Mackie b
a VTT Technical Research Centre of Finland,P.O.Box 1000,FI-02044VTT,Espoo,Finland b
Institute of Food Research,Norwich Research Park,Norwich NR47UA,UK
a r t i c l e i n f o
Article history:
Received 2July 2011
Accepted 21November 2011Keywords:
b -Lactoglobulin Tyrosinase
Transglutaminase Cross-linking Interface
a b s t r a c t
Effective and controlled use of cross-linking enzymes in structure engineering of food systems depends on characterization of the favorable conditions for enzyme-substrate complex and the limiting factors for the desired modi ?cation.In this respect,we analyzed the susceptibility of bovine b -lactoglobulin (BLG)to enzymatic cross-linking by Trichoderma reesei tyrosinase (TrTyr)and transglutaminase (TG).Changes in BLG molecular structure were determined at pH 6.8,7.5and 9.0before and after high-temperature heat treatment.The conformational change was linked to ef ?ciency of protein cross-linking.BLG was not susceptible to TrTyr without heat treatment.TG,however,induced inter-molecular cross-links at pH 7.5and 9.0.After the heat treatments,BLG molecules adopted a molten-globule-like conformation.Both of the enzymes were able to form inter-molecular cross-links between heat-denatured BLG molecules.Electrophoretic mobility and broadness of the oligomer bands created by both enzymes on SDS-PAGE gels showed differences which were linked to the availability and number of target amino acid resi-dues.Evidence for intra-molecular cross-linking was obtained.Once adsorbed to air/water interface,BLG formed a viscoelastic surface ?lm which was characterized by surface shear rheology.Application of cross-linking enzymes under a dense layer of BLG molecules at the interface led to decreasing G 0with time.Intra-molecular links were most probably favored against inter-molecular on packed BLG layer leading to constrained molecules.Results in general emphasize the importance of structural and colloidal aspects of protein molecules in controlling inter/intra-molecular bond formation by cross-linking enzymes.
ó2011Elsevier Ltd.All rights reserved.
1.Introduction
Enzymatic cross-linking of food proteins has created increasing interest in recent years,the main argument being that the intro-duction of covalent links modi ?es the structure of the protein networks and that;due to the speci ?city of cross-linking enzymes,the properties of the structure formed can be controlled.In order to make ef ?cient use of enzymes in food material modi ?cation,it is important to understand the availability of substrate protein for catalysis,i.e.,the accessibility of reactive residues and also the probability of side-reactions.The various physical factors which are of importance in this respect are presented in Scheme 1.It has already been demonstrated that proteins without a con ?ned tertiary structure are better substrates for cross-linking enzymes compared to globular proteins in their native form (Hellman,
Mattinen,Fu,Buchert,&Permi,2011;Mattinen et al.,2006;Mattinen,Lantto,Selinheimo,Kruus,&Buchert,2008).
The most abundant of whey proteins in bovine milk is b -lacto-globulin (BLG),which has a well-characterized globular structure.Under physiological conditions,bovine BLG exists as a homodimer consisting of w 18,3kDa monomeric subunits.Each BLG monomer has 162amino acids containing one free cysteine and two disul ?de bridges folded in a calyx shaped b -barrel structure.The free cysteine remains buried inside the molecule in native state and becomes exposed and reactive only after certain changes in tertiary structure (Brownlow et al.,1997;Considine,Patel,Anema,Singh,&Creamer,2007;Qin et al.,1998).In its native state,BLG is resistant to enzymatic reactions.Its resistance to peptic enzymes,for example,is stated to be due to a highly stable,compact tertiary structure which hinders most of the speci ?c peptides for cleavage (Reddy,Kella,&Kinsella,1988).Similarly,enzymatic cross-linking of native BLG is limited possibly due to the hindrance of target amino acid side chains for cross-linking enzymes.In fact,complete
*Corresponding author.Tel.:t358400157616;fax:t358207227071.E-mail address:dilek.ercili@vtt.?(D.
Ercili-Cura).Contents lists available at SciVerse ScienceDirect
Food Hydrocolloids
journal homepa ge:
https://www.sodocs.net/doc/945904575.html,/locate/foodhyd
0268-005X/$e see front matter ó2011Elsevier Ltd.All rights reserved.doi:10.1016/j.foodhyd.2011.11.010
Food Hydrocolloids 28(2012)1e 9
or partial denaturation of the molecule by physical or chemical means has been reported to increase the extent of protein cross-linking (Eissa,Puhl,Kadla,&Khan,2006;Eissa,Satisha,&Khan,2004;F?rgemand,Murray,&Dickinson,1997;Faegermand,Otte,&Qvist,1998).
Exposure to different pH conditions,heat treatments,and adsorption to interfaces are all potential ways to induce conforma-tional changes,which could then increase accessibility of BLG for enzymatic catalysis.In the region of Tanford transition,which is centered around pH 7.5,BLG molecule attains a molten-globule-like structure i.e.,the protein retains its secondary structure and shows a compact tertiary structure but with increased mobility and looser packing thus increased reactivity (Tanford,Bunville,&Nozaki,1959;Taulier &Chakilian,2001;Yang,Dunker,Powers,Clark,&Swanson,2001).At alkaline pH,BLG undergoes irreversible denaturation which induces monomer-aggregate equilibrium depending on protein concentration,ionic strength and temperature (Barteri,Gaudiano,Rotella,Benagiano,&Pala,2000;Townend,Herskovits,Timasheff,&Gorbunoff,1969).De Wit (2008)reviewed the thermal behavior of bovine BLG at temperatures up to 150 C.Accordingly,between 60and 70 C (pH >6.8)partial unfolding of BLG to molten-globule-like state and irreversible (to a limited extent)modi ?cation of monomers induced by exposed thiols were reported.At higher temperatures,increased unfolding followed by aggregation due to disul ?de formation and hydrophobic interactions take https://www.sodocs.net/doc/945904575.html,plete unfolding of BLG by breakdown of disul ?de bonds can be achieved only after heating to temperatures as high as 125 C (Watanabe &Klostermeyer,1976).Proteins are surface active in nature and it has long been hypothesized that once they adsorb to interfaces,they partially unfold and arrange their conformation such that hydrophobic patches align with the hydrophobic phase (Wilde,2000).It is dif ?cult to assess the exact conformation of adsorbed BLG molecules due to technical limitations in analysis methods,yet several studies have shown that the change in secondary structure is highly limited (Martin,Meinders,Bos,Cohen Stuart,&van Vliet,2003;Meinders &de Jongh,2002),and no decisive conclusions have yet been reported on tertiary structure.
Transglutaminase (TG,EC 2.3.2.13,g -glutamyl-peptide,amine-g -glutamyl transferase)is the most extensively studied protein cross-linking enzyme and has been thoroughly reviewed for its effects on dairy systems speci ?cally (Jaros,Partschefeld,Henle,&Rohm,2006).TG cross-links peptides and proteins through an acyl transfer mechanism between glutamine and lysine residues.In
the absence of amines,water serves as an acyl acceptor leading to conversion of glutamines to glutamic acid (deamidation)(Grif ?n,Casadio,&Bergamini,2002;Kashiwagi et al.,2002).Oxidative enzymes as cross-linking enzymes are also gaining wider attention for the ability to oxidize protein substrates and subsequently modify the structural (Ercili-Cura et al.,2009,2010;Lantto,Puolanne,Kalkkinen,Buchert,&Autio,2005;Lantto,Puolanne,Kruus,Buchert,&Autio,2007;Selinheimo,Autio,Kruus,&Buchert,2007)and nutritional (Monogioudi et al.,2011;Stanic et al.,2010;Tantoush et al.,2011)properties of food products.Tyrosinases (EC 1.14.18.1)are mono-oxygenases which catalyze ortho -hydroxylation of monophenols to o -diphenols,which are further oxidized to o -quinones (Solomon,Sundaram,&Machonkin,1996).Quinones are highly reactive and can further react non-enzymatically with each other or with thiol and/or amino groups resulting in formation of di-tyrosine,tyrosine e cysteine and tyro-sine e lysine cross-links in protein substrates (Burzio,Burzio,Pardo,&Burzio,2000;Ito,Kato,Shinpo,&Fujita,1984;Matheis &Whitaker,1984).Thus,modi ?cation of BLG molecule by tyrosi-nase depends mainly on the accessibility of tyrosine residues.
While the chemistry of enzymatically created cross-links is mainly known and a vast number of studies describe the effect of cross-linking on the macroscopic structure of a protein matrix (Buchert et al.,2010),relatively little is known about the physical constraints preceding enzymatic catalysis and the consequences of the links formed within the substrate molecules.The aim of this study was to relate pH-and temperature-induced conformational changes of BLG and constraints given by the environment,such as in solution vs.adsorbed layer,on its quality as a substrate for tyrosinase and transglutaminase catalyzed reactions.2.Materials and methods 2.1.Preparation of BLG solutions
Bovine BLG (90%by PAGE,mixture of A and B variants)was purchased from Sigma (St.Louis,MO,USA)and used without further puri ?cation.Solutions of BLG were prepared at concentra-tion of 1or 2mg mL à1at three different pH values;6.8,7.5and 9.0.For pH 6.8and 7.5,sodium phosphate buffer (20mM)and for pH 9.0,borate buffer (20mM)were used in all experiments.After dissolving,BLG solutions were left mixing for 1h for equilibration.When needed,solutions were heat treated at 80 C for 30min (in water bath)or at 125 C for 2min (in a block heater).After the heat treatment,samples were directly put onto ice for 5min and after-ward tempered at room temperature.Before the analysis (CD or interfacial rheology),solutions were further diluted to desired concentrations and left at room temperature for 30e 60min for equilibration.
2.2.Circular dichroism (CD)
Changes in secondary and tertiary structure of BLG subjected to different pH conditions and heat treatments were assessed by CD.Spectra were recorded using a CD spectropolarimeter (JASCO J710,Jasco Ltd.,Japan).Far-UV (180e 260nm)and near-UV (240e 330nm)CD spectra were recorded at room temperature with cells of 0.5mm and 10mm path lengths,respectively.Sample concentrations used for far-and near-UV were 0.2mg mL à1and 2mg mL à1respectively.Four accumulations were collected at 20nm min à1scan speed,1nm bandwidth,0.5nm data pitch with 2s response time during far-UV measurements.For near-UV,six accumulations were collected at 50nm min à1scan speed,1nm bandwidth,0.5nm data pitch with 1s response time.The instrument was calibrated using ammonium camphorsulfonic acid.Buffer baseline was
subtracted
Scheme 1.Representation of conditions required for enzymatic cross-linking of proteins.
D.Ercili-Cura et al./Food Hydrocolloids 28(2012)1e 9
2
from each spectrum.The far-UV unsmoothed spectra are repre-sented as molar CD[deg cm2dmolà1]based on the mean residue molecular weight.The near-UV CD spectra are presented in measured ellipticities[mdeg]without smoothing.
2.3.Enzymatic modi?cation
Trichoderma reesei tyrosinase(TrTyr)was produced at VTT as described by Selinheimo et al.(2006).Tyrosinase activity was measured spectrophotometrically by using15mM L-DOPA(Sigma, St.Luis,MO,USA)as substrate at pH7.0according to Robb(1984). The commercial Ca2t-independant TG preparation(ActivaòWM, Ajinomoto Inc.,Japan)was supplied by Vesantti Oy(Helsinki,Fin-land)and further puri?ed at VTT as described in Lantto et al.(2005). The activity of the TG preparation was measured by using0.03M N-carbobenzoxy-L-glutaminyl-glysine as substrate at pH6.0accord-ing to Folk(1970).Accordingly,the speci?c activity of TrTyr was measured to be425nkat mgà1and of TG was500nkat mgà1. Enzymes were dosed based on the substrate protein concentration, i.e.nkat per g of protein in solution(nkat gà1).Heat treated and untreated BLG solutions(1mg mLà1)were mixed with enzyme preparations at the dosage of1000nkat gà1and incubated for18h under constant stirring either at room temperature or40 C respectively for TrTyr and TG.For TrTyr reactions,oxygen was continuously supplied by leaving the caps open under a closed container to limit evaporation.
2.4.SDS-PAGE analysis
The extent of BLG oligomerization caused by disul?de bridging was analyzed by non-reducing SDS-PAGE.Aliquots(65m L)of sample solutions(1mg mLà1)were mixed with25m L of LDS sample buffer(glycerol,tris base,tris e HCl,lithium dodecyl-sulfate (LDS),EDTA,serva blue G250,phenol red)(4?)(Invitrogen Ltd., Paisley,UK)and10m L of distilled water for non-reducing PAGE. Enzyme-induced cross-linking of BLG was analyzed by reducing SDS-PAGE.Aliquots of enzyme-free control and enzyme-treated samples were mixed with25m L of LDS buffer and10m L of50mM DTT(Amersham Biosciences,Uppsala,Sweden)solution.Samples were heat treated at70 C for10min before loading to the gel.Non-reducing and reducing SDS-PAGE samples were loaded on12%Bis-Tris gels(NuPAGE,Invitrogen Ltd.,Paisley,UK)and run according to producer’s protocol by using MOPS running buffer(20?)(Invi-trogen Ltd.,Paisley,UK).Gels were stained using coomassie(Sim-plyBlue SafeStain,Invitrogen Ltd.,Paisley,UK).Mark12?unstained protein molecular weight standard(Invitrogen Ltd.,Paisley,UK) was used in reducing SDS-PAGE gels.No molecular weight standard was shown in non-reducing page gel,however,the monomer and dimer bands were con?rmed on another gel(data not shown).
2.5.Interfacial rheology
The surface shear rheological properties of BLG at the air e water interface were measured using a stress-controlled rheometer(AR-G2,TA Instruments,U.K.)equipped with a Pt e Ir du Noüy ring (13mm diameter).The ring was?amed prior to each experiment. BLG stock solution(1mg mLà1),prepared at pH6.8was used with or without heat treatment(80 C,30min).Stock solution was diluted1:20(0.05mg mLà1)in the same buffer(20mM sodium phosphate at pH6.8)before the rheological measurements.The instrument was mapped by performing rotational and oscillatory mappings prior to each measurement.Exactly50mL of prepared BLG solution was placed in a glass dish of60mm diameter and the du Noüy ring was placed onto the surface according to the manu-facturer’s instructions.Surface shear moduli were followed at21 C at0.1Hz and constant strain of0.5%,which was measured to be in the linear viscoelastic region(moduli were not dependent on the applied strain).The evolution of the moduli due to adsorption of BLG molecules were recorded during a1h time sweep.After that, the measurement was paused for10min and the prepared enzyme dilutions providing a dosage of10000nkat gà1(or50000nkat gà1 for TG at non-heat-treated condition)for both TrTyr and TG were injected by using a50m L Hamilton glass injector to the subphase in small portions from several points.Enzyme dosage was increased to10000nkat gà1in interfacial measurements to compensate the effect of dilution on catalytic activity.Even if the local concentration of the substrate is high at the interface,no such gradient is expected in enzyme concentration.At the end of10min,another time sweep of8h was performed using the same parameters.Enzyme injection caused a reversible disturbance in BLG?lms as was observed in moduli evolution in time.To retard the evaporation,the measure-ment area was covered by the shield provided by the producer with several beakers of water inside and the open sides were sealed carefully with para?lm.The surface of the sample dish was also covered with a lid which does not disturb the measurement.
The interfacial shear rheological methods generally do not have good reproducibility(Kr?gel&Derkatch,2010).However,qualita-tive data can be achieved.As explained above,the protein adsorption and?lm formation was followed for1h for BLG alone before injection of the enzyme in each measurement.That way, a representative?lm was formed and the effect of any enzymatic action could be assessed qualitatively.Each measurement was replicated at least twice and the same trends were repeatedly observed upon enzyme treatment of BLG?lms.
2.6.Surface pressure measurements
The surface pressure of adsorbed BLG layers was measured with a KSV?lm balance(Minimicro series,KSV Instruments)using a platinum Wilhelmy plate at room temperature.The aim here was to create an adsorbed protein layer in the same conditions as in the rheological measurements.The same dish that was used for rheology measurements(60mm diameter,glass)was?rst?lled with45g of pure water at room temperature.The Wilhelmy plate (washed with ethanol,?amed and wetted prior to each experi-ment)was dipped into the water and the balance was zeroed at surface tension of around72mN mà1.After this,the water was removed from the dish and the same amount of(45g)BLG solution (0.05mg mLà1)prepared in the appropriate buffer was placed.The position of the Wilhelmy plate was not changed,thus the Wilhelmy plate was dipped to the same height in to the BLG solution as it was dipped in water.The surface pressure measurements started as soon as the BLG solution was in the dish and touched the Wilhelmy plate.Data was recorded in60sec intervals.After1h of adsorption, 45m L of enzyme dilution,or only buffer for the control sample,was injected to the subphase in the same manner as for the rheological measurements.Surface pressure data acquisition was continued during enzyme injection,which again caused a reversible disrup-tion in surface pressure.After the enzyme injection,measurements continued for8h.The experiments could be run for9h with negligible evaporation as the cabinet was sealed and several beakers?lled with water were placed inside the cabinet.
3.Results
3.1.BLG conformation at different conditions
BLG conformation was analyzed at three different pH values:6.8, 7.5and9.0,at which the solutions were either not heated or heated to80 C(30min.)or125 C(2min.).The lowest pH,6.8was chosen
D.Ercili-Cura et al./Food Hydrocolloids28(2012)1e93
as a reference as it is the physiological pH in which BLG is found
naturally(the molecule is in the initial phases of Tanford transition at
pH6.8.We will still refer this state as‘native’throughout the text).
The higher pH values;pH7.5and9.0were chosen as they represent
the Tanford transition(Tanford et al.,1959)and the start of irre-
versible denaturation(Barteri et al.,2000;Townend et al.,1969),
respectively.Far-UV spectra of the non-heated and heat-treated BLG
solutions are shown in Fig.1.At room temperature,far-UV spectra of
BLG at both pH6.8and7.5showed a wide minimum at around
216nm,typical of a predominantly b-sheet structure(Fig.1A).At pH 9.0,a shift in negative peak was observed toward the region around
210nm.Upon heating to80 C and subsequent cooling,the
minimum peak was further shifted to the region below205nm at all
pH values to the same extent(Fig.1B).The same phenomena were
observed upon heating at125 C although with a more pronounced
effect at pH9.0(Fig.1C).The CD spectrum of a random coil shows
a single intense negative peak near200nm(Kelly,Jess,&Price,
2005).Observed effect of heat treatment on secondary structure is
in accordance with other authors who reported loss of a-helices and b-sheets and increase in random coil structures upon heat treatment
of BLG(Carrotta,Bauer,Waninge,&Rischel,2001;Kim,Cornec,&
Narsimhan,2005).The effect of heat treatments for different pH
values are depicted in Fig.1D e F.At all pH values,the negative peak at w205nm was similar for both80 C and125 C heat treatments indicating no further loss of secondary structure upon125 C treat-ment as opposed to DSC data previously reported by De Wit(1981).
Near-UV CD spectra of BLG solution at the same pH conditions
with/without heat treatment at80 C(30min.)were also followed (Fig.2).Near-UV CD spectra of BLG at both pH6.8and7.5showed very intense negative peaks at293and285nm which are the typical features present in native folded conformation.No signi?-cant difference was observed between pH6.8and7.5in non-heat-treated samples which is in accordance with Taulier and Chakilian (2001).At pH9.0,the intensity of negative bands at285and 293nm were decreased compared to neutral pH in the non-heat-treated samples.This indicates a decrease in tertiary structure in the region of at least one of the two tryptophan residues.Upon heat treatment,those bands were signi?cantly diminished at all pH values,with more signi?cant change at pH9.0(Fig.2).Lower negative ellipticity values were obtained at all wavelengths for heat-treated samples.The change was signi?cant also between260 and280nm.The loss of negative ellipticity in the near-UV CD spectra indicates a signi?cant loss of tertiary structure.However,it should be kept in mind that there was2e3h in between the heat treatments and the CD analysis.Some extent of re-folding could have occurred during this period.
3.2.Oligomerization of BLG caused by inter-molecular disul?de bond formation
The degree of BLG oligomerization due to formation of inter-molecular disul?de bonds was analyzed by non-reducing SDS-PAGE(Fig.3).Without any heat treatment,intense bands of BLG monomers and faint bands of BLG dimers are visible at all three pH values.This unusual dimer band(SDS-resistant dimer)was previ-ously reported and was attributed to the BLG covalent dimers
that
Fig.1.Far-UV CD spectra of non-heated and heat-treated BLG molecules.Effect of pH at different heat treatments(A,B,C);effect of heating at different pH values(D,E,F).(D,E,and F are re-organization of the same data in A,B,C).The legends for the?rst and the second columns are shown in graphs A and D,respectively.
D.Ercili-Cura et al./Food Hydrocolloids28(2012)1e9
4
are naturally formed during processing and storage of such protein powders(de Jongh,Gr?neveld,&de Groot,2001;Muhammad, Croguennec,Julien,Michel,&Sa?d,2009).The?gure shows that without heat treatment there were no disul?de-stabilized oligo-mers observed at pH6.8and7.5.At pH9,a slight smear could be seen around the SDS-resistant BLG dimer band.Exceptionally at pH 9.0a faint band around14kDa is also visible(Fig.3)which might be due to alkaline autolysis of BLG molecule(Christensen,1949).After heat treatment,disul?de linked dimers,trimers and even bigger oligomers at both pH6.8and7.5for both80 C and125 C treat-ments were observed.However,at pH9.0,only dimers were formed and to a relatively low extent for both heat treatments. There were no differences between the two heat treatments with respect to disul?de bond formation(Fig.3).The majority of BLG remained monomeric in all cases.
3.3.Enzymatic cross-linking of BLG
For cross-linking reactions,BLG was incubated with TrTyr for 18h at room temperature and with TG for18h at40 C.Room temperature was chosen for TrTyr as the enzyme is not very ther-mostable.It has a half-life of15min at50 C,and18h at30 C. TrTyr has its highest activity and stability at neutral and alkaline pH values with the optimum at pH9(Selinheimo et al.,2006).TG is reported to be stable at40 C which is also in the range of its optimum temperature.TG is stable at a pH range of5.0e7.0with an optimum at pH6.0,but signi?cantly loses activity outside of this pH range(Lu,Zhou,Tian,Li,&Chen,2003).
Enzymatic cross-linking of BLG was analyzed by SDS-PAGE in reducing conditions.Enzymatic catalysis at different pH conditions (without heat treatment)is shown in Fig.4.Without any heat treatment or enzyme addition,BLG monomers appear around 18kDa together with a faint band at around36kDa,which belongs to the SDS-resistant dimer.Once more,a faint band at low molec-ular weight(14kDa)is visible at pH9.0(Fig.4).The TrTyr-treated BLG samples show no difference compared to control samples at pH6.8and7.5.At pH9.0only faint smears below and above the SDS-resistant BLG dimer band could be observed after TrTyr treatment When BLG was treated with TG at pH6.8no cross-linking was observed.However,at pH7.5oligomer bands which are scattered below and above36kDa are visible.Besides high molecular weight smears,a clear band below the BLG monomer at around14kDa was also formed.Similarly,at pH9.0,a smeary band was visible below36kDa when treated with TG.Unlike pH7.5,at pH9.0no smearing was observed above36kDa.Moreover,the band around14kDa was intensi?ed and smeared upwards at pH 9.0compared to the one at pH7.5in TG-treated samples(Fig.4).
After the heat treatments at80 C or125 C,both TrTyr and TG treatments resulted in formation of covalent cross-links at all three pH values(Fig.5A and B).The?rst three lanes of each gel in Fig.5 represent the heat-treated controls(enzyme-free).No covalent oligomerization was observed without the presence of enzymes at any pH.Firstly,there were no differences in the patterns of olig-omer formation due to either of the enzymes when comparing two heat treatment conditions.Therefore,Fig.5A and B are analyzed as one.After heat treatment,TrTyr treatment at1000nkat gà1resul-ted in the formation of dimers,trimers and higher molecular weight oligomers at both pH6.8and7.5(Fig.5A and B).At pH9.0, degree of TrTyr cross-linking was decreased as some of the high molecular weight bands(>97kDa)were missing or fainter compared to those at pH6.8and7.5.Similarly,TG treatment at 1000nkat gà1also resulted in protein cross-linking depicted by formation of intense bands at the high molecular weight range and a clear loss of intensity of the BLG monomers(Fig.5A and B). However,the bands created by TG action were not exact but broad bands scattered signi?cantly downwards on the gel,which was actually the case also in the TG-cross-linking products in the solution without heat treatment(Fig.4).Dimers were observed
as
Fig.3.Non-reducing SDS-PAGE of BLG at pH6.8,7.5and9.0;heat treated at80 C
(30min)and125 C(2min)
separately.
Fig.4.Reducing SDS-PAGE of BLG at pH6.8,7.5and9.0without any heat treatment.
BLG was treated by TrTyr(1000nkat gà1)and TG(1000nkat gà1)for18h at RT and
40 C,respectively.The last two lanes are TrTyr and TG loaded
alone.
Fig.2.Near-UV CD spectra of non-heated(dashed lines)and heat treated(solid lines)
BLG molecules at pH6.8(black),pH7.5(gray),and pH9.0(light gray).
D.Ercili-Cura et al./Food Hydrocolloids28(2012)1e95
broad,smeared bands with an increased mobility compared to the original BLG dimer.They were identi ?ed at around 29kDa instead of 36kDa.The same phenomenon was observed for TG-induced trimers and higher oligomers.When the reaction was carried out at pH 9.0,TG-induced cross-linking was signi ?cantly limited compared to pH 6.8and 7.5.Only a smeared dimer band again with an increased mobility (around 32kDa)was observed.Besides the formation of higher molecular weight bands,in every lane that was treated by the enzymes,there were also low molecular weight bands which appear below the BLG monomer bands.More specif-ically,formation of two exact bands between 14kDa and 18kDa for the case of TrTyr and one exact band at 14kDa for TG were observed at pH 6.8and 7.5after both heat treatments.At pH 9.0,those low molecular weight bands were rather broad and intensi-?ed in TG-treated samples (Fig.5A and B).
The enzymes were also run separately on SDS-PAGE gels (Figs.4and 5,last two lanes).At the studied enzyme dosage,no bands due to the enzyme proteins were visible in sample lanes.3.4.Interfacial properties
Interfacial shear rheological properties of both non-heat-treated and heat-treated BLG were measured.The aim was to analyze the enzymatic modi ?cation of BLG at the interface indirectly through the viscoelastic properties of the interfacial ?lm.The results ob-tained by TG treatment only were shown as the results obtained by TrTyr were similar to TG.
BLG molecules adsorbed rather quickly to the air/water interface giving an elastic-dominant response (loss tangent (G 00/G 0)<1,not shown)immediately after the start of the measurements (Fig.6).The initial surface shear elastic modulus (G 0)was around 0.020N m à1at both non-heat-treated and heat-treated conditions (data not shown).The adsorption of BLG molecules were followed for 60min until a representative ?lm was formed.At the end of 60min,enzyme or only buffer (for the control samples)were injected and the second time sweeps were started.The protein ?lm was reversibly ruptured during the injections,as shown by a decrease in G 0values in the initial phase of the second time sweeps which recovers in short time.In the case of the buffer injected controls,the ?lm strength continued to grow until a ?nal G 0value that was w 1.2times higher compared to the value that was attained at 60min.In the case of non-heat-treated BLG,injection of TG at the dosage of 10000nkat g à1did not cause a considerable effect.The G 0values leveled at a lower ?nal G 0compared to the
control,but not different compared to the 60min value which was right before the enzyme injection.When the dosage of injected TG was increased to 50000nkat g à1,a considerable (w 23%)decrease in ?lm strength was observed compared to the 60min value (Fig.6A).
Upon injection of 10000nkat g à1TG to the ?lm formed by heat-treated BLG,G 0continued to rise initially which was followed
by
Fig.5.Reducing SDS-PAGE of BLG prepared at pH 6.8,7.5and 9.0that was heat treated at 80 C,30min.(A)and heat treated at 125 C,2min.(B).BLG was cross-linked by TrTyr (1000nkat g à1)and TG (1000nkat g à1)for 18h at RT and 40 C,respectively.The last two lanes,are TrTyr and TG loaded
alone.
Fig.6.Changes in interfacial shear elastic modulus (G 0)in time for non-heated (A);and heat treated (80 C,30min)(B)BLG solutions at pH 6.8.BLG adsorption was followed for 1h,after which 10000nkat g à1transglutaminase (TG)was injected to the subphase (B ).Only buffer was injected to the control samples (C ).In addition,a curve with 50000nkat g à1TG injection (,)was shown in (A),and a curve with inactivated TG injection (-)was shown in (B).Vertical bars represent standard deviation.Values were normalized to the G 0values at 60min (right before enzyme or buffer injection)for each sample.
D.Ercili-Cura et al./Food Hydrocolloids 28(2012)1e 9
6
a noticeable decrease after 150e 200min of measurement time.The G 0of TG-treated sample was decreased (w 13%)at 550min compared to the value at 60min (Fig.6B).When the BLG molecules were more enzyme-susceptible (heat-treated case),the effect re-?ected on the interfacial shear elastic modulus was higher compared to the more native state (non-heat-treated case)for the same enzyme dosage.Overall,there was a negative effect of the enzyme-treatments on development of G 0which was more evident when BLG was heat treated.The probable effect of adsorption of enzyme protein itself was also tested by conducting the experi-ments in the same manner but using inactivated enzyme prepa-rations.TG was inactivated by incubation at 50 C for 18h.No considerable effect due to adsorption of the TG proteins or any contaminating surface active component was detected (Fig.6B).
To obtain more insight on the adsorbed ?lms before and after the enzyme injections,the saturation of the air e water interface was followed by surface pressure measurements conducted in parallel with the rheological measurements.Results representative of the samples in Fig.6B are depicted in Fig.7.The initial surface pressure values were already very high (w 20mN m à1)and did not change much in the course of 9h.The disruption of the ?lm caused by enzyme or buffer injections was quickly recovered for all samples and no signi ?cant effect of the enzyme protein added to the solution was re ?ected to surface pressure (Fig.7).4.Discussion
As BLG is rather resistant to enzymatic modi ?cation in the native state,an attempt was made to relate conformational changes induced by pH and heating to its susceptibility toward trans-glutaminase and tyrosinase.Attention is given to the physical constraints in formation of intra-and/or inter-molecular links,either caused by protein conformation or by its environment,bulk or interfacial systems.
The far-UV results showed b -sheet-dominant structures at all pH values studied (without heat treatment)with a slight change only at pH 9.0.In near-UV spectra,the intensity of minimum peaks was decreased at pH 9.0compared to neutral pH indicating that the speci ?c and rigid packing of aromatic residues,namely tryptophan and tyrosine,was partly lost at pH 9.0.Interestingly,TG-induced cross-linking was possible at pH 7.5without any heat treatment (Fig.4),and was thus not linked with the minor changes in secondary or tertiary structures observed only at pH 9.0.The only
conformational change in BLG at around pH 7.5that could explain this behavior is a local displacement of the EF loop opening the calyx interior (Qin et al.,1998)and the shift in monomer e dimer equilibrium toward monomeric form.Neither of these can be detected by far-or near-UV CD (Taulier &Chakilian,2001).At pH 7.5,reactivity of 4glutamines and 3lysines in BLG has been re-ported previously (Nieuwenhuizen,Dekker,Gr?neveld,de Koster,&de Jong,2004),but as none of these identi ?ed residues can be directly associated with the dimer interface or the EF loop,it is not evident why they become reactive.At pH 9.0,inter-molecular cross-linking with TG was limited to smeared dimers only.The reason could well be the limited activity of TG at pH 9.0(Lu et al.,2003).
Tyrosinase,which is still active at pH 9.0,was unable to catalyze cross-linking at any of the pH values in unheated BLG.This was most likely because there are only four tyrosine residues in BLG.Two of the tyrosines have been shown to be close to the surface and thus exposed in native state but still somewhat hindered and two tyrosines are buried (Brownlow et al.,1997;Townend et al.,1969).The loosening of the three-dimensional structure and increased backbone ?exibility of BLG molecule at pH 9.0has previously been associated with increased reactivity toward tyrosinase (Partanen et al.,2011).Compared to the present study,a higher substrate concentration was used by Partanen et al.,which could well indi-cate the co-existence of a minor fraction of reactive conformation and a major non-accessible fraction.
Upon heat treatments,a similar decrease in ordered secondary structure with increase in random coil in far-UV spectra and a signi ?cant loss of tertiary structure around the tyrosine and tryptophan residues in the near-UV spectra was found,which enabled cross-linking of BLG by both enzymes.Cross-linking of BLG upon loss of native structure is in agreement with previous reports showing the need to denature either by means of heat (Eissa et al.,2006;Sharma,Lorenzen,&Qvist,2001),high pressure (Lauber,Krause,Klostermeyer,&Henle,2003)or chemical agents (Eissa et al.,2006;F?rgemand et al.,1997,1998)for signi ?cant suscepti-bility of BLG to cross-linking.After heat treatment,there was no difference between pH values 6.8and 7.5in the patterns of formed oligomers by either of the enzymes,but pH 9.0was clearly least ef ?cient in catalyzing formation of inter-molecular cross-links.This could be mainly due to the effect of pH in activity and stability of the enzymes:pH 9.0was reported to be the optimum pH for TrTyr (Selinheimo et al.,2006),but there is not much data about its stability at that pH.And for TG,pH 9.0is rather high as was also discussed for the non-heat-treated case.
An interesting outcome of the SDS-PAGE patterns of heat-treated BLG was the difference observed between the shape and mobility of bands after incubation with TrTyr and TG (Fig.5).The broadening and increased mobility of the TG-induced oligomer bands could be attributed to heterogeneity of the created covalent bonds between and within the related BLG molecules due to higher number of reactive residues for TG as compared with TrTyr.It should also be noted that in the absence of available lysines in close proximity of glutamines,TG action induces deamidation reaction.As a consequence,glutamines are transformed into glutamic acid residues,which in turn lower the pI of BLG (Nieuwenhuizen et al.,2004)causing more heterogeneity.Another explanation for increased band mobility could be extensive levels of intra-molecular cross-linking which might affect overall shape of the SDS-denatured molecule.Hellman et al.(2011)have recently shown that a model globular protein was not inter-molecularly cross-linked by tyrosinase unless partially denatured (or unless having water accessible tyrosine close to a nucleophilic amino acid in the folded form).However,intra-molecular cross-linking was still prevalent which induced formation of non-native
monomers
Fig.7.Changes in surface pressure for heat-treated BLG solution at pH 6.8.Adsorption was followed for 1h,until the point of ‘enzyme injection ’at which 10000nkat g à1of TrTyr (gray)or TG (light gray)was injected to the subphase.Only buffer was injected to the control sample (black).Vertical bars represent standard deviation.
D.Ercili-Cura et al./Food Hydrocolloids 28(2012)1e 97
which showed delayed elution in size exclusion chromatography. Accordingly,we suggest that the low molecular weight bands observed below the BLG monomers(w14kDa)could as well belong to intra-linked,or alternatively,in the case of TG,deamidated monomers.The fact that the low molecular weight bands were intensi?ed at pH9.0could be due to physical constraints of BLG molecule at that pH.For example,lower amounts of disul?de linked dimers were observed at pH9.0compared to other pH values which might favor internal cross-linking of monomeric subunits. Finally,those low molecular weight bands are not caused by protease contaminations in enzyme preparations as both TrTyr and TG were tested negative for proteolytic activity at neutral pH(data not shown).
Interfacial shear rheology is a good approach to analyze the intra-and/or inter-molecular interactions at interfaces(Kr?gel& Derkatch,2010).Our driving idea for using enzymatic cross-linking at an adsorbed layer of BLG was the reported behavior of BLG to partially unfold once adsorbed to air/water interface(see reviews by Bos&van Vliet,2001;Murray,2002;Wilde,2000). Many reports have been published on unfolding kinetics of globular proteins at interfaces with a common conclusion that partial unfolding upon self-re-arrangement of adsorbed proteins may happen if the protein has a certain minimum area to expand. Surface pressure measurements revealed that at the BLG concen-tration studied,adsorption of protein molecules to the surface was rather fast.In fact,the surface pressure reached w20mN mà1 within seconds.Even though the interface was saturated with BLG molecules instantly,G0continued to increase with time.That implies on-going structural organization with increasing lateral interactions between adsorbed molecules.Enzymes were injected underneath the packed protein layer.After addition of TG to the subphase,?rstly the?lm recovered from the injection damage similar to control,but in time,?lm strength was lowered as was observed by decreasing G0.We would expect that creation of inter-molecular covalent bonds by the enzymes would increase the?lm strength.In fact,F?rgemand and Murray(1998)have reported increased surface dilatational elastic modulus upon TG treatment of adsorbed BLG molecules.On the other hand,Romoscanu and Mezzenga(2005)showed that glutaraldehyde-induced cross-link-ing increased the elastic modulus of non-densi?ed BLG interface while the effect was reversed when glutaraldehyde was applied on densi?ed,folded interface.Accordingly,we may claim that,once adsorbed fast at such high surface concentration,BLG molecules attained a constrained structure which limited formation of enzyme-induced inter-molecular covalent links.However,intra-molecular links could well be formed within the adsorbed mole-cules.Hellman et al.(2011)have recently shown that intra-molecularly cross-linked proteins are locked in their globular fold. Such bonds created by both TrTyr and TG would then further impede the re-arrangement of protein adsorbed to the interface during aging which would lead to diminishing of physical pro-tein e protein interactions at the interface.Eissa et al.(2006)re-ported that formation of compact molecules by TG action limits exposure of hydrophobic regions thus attenuates hydrophobic interactions in whey proteins which might similarly limit further development of hydrophobic interactions between the adsorbed proteins and lead to decreasing?lm strength.Another argument for TG action could be the deamidation which would increase the negative charge of the protein molecules leading to weakening of inter-molecular interactions as a result of increased electrostatic repulsion.
There are several techniques used for detection of the confor-mation of adsorbed proteins but these are still being developed.So far,the evidence for change of tertiary structure was most frequently obtained through indirect methods such as following the change in surface pressure,surface viscosity or viscoelasticity in time which would infer evidence for inter-molecular interactions and a change of fold.Interpretation of the indirect evidence attained depends,however,on two assumptions as was made clear by Wierenga and Gruppen(2010)recently:1.To consider proteins as?exible polymer chains that adopt different structures depend-ing on the surface load or pressure,2.To consider proteins as colloidal particles similar to hard spheres which preserve structural integrity upon adsorption.Accordingly,it is challenging to fully understand what gives the elastic response in such systems and how the?lm strength can be tailored for e.g.improved foam stability.
5.Conclusions
Subjecting BLG to slightly alkaline(pH7.5)and alkaline(pH9.0) pH conditions led to enzymatic cross-linking by transglutaminase. This limited cross-linking could be due to a minor population shifting toward a molten-globule state,as little change in secondary or tertiary structures was found at pH7.5.Upon heat treatment and cooling,formation of covalently linked BLG oligomers was associ-ated with molten-globule-like conformation of the substrate which shows as a small change in secondary structure and a signi?cantly disturbed tertiary structure.As with monomers,increased mobility of transglutaminase-induced oligomers on gel electrophoresis is suggested to be due to extensive internal cross-linking.The smearing of the bands is explained by the higher number of reac-tive residues for TG as compared with TrTyr,and therefore an increased number of possible combinations of inter-molecular cross-links leading to formation of heterogeneous oligomers. When heat-treated BLG molecules adsorbed to air/water interface were enzyme-treated,the shear elastic modulus decreased with time.We suggest that due to rapid saturation of the surface,the mobility of the BLG becomes a limiting factor for inter-molecular cross-linking.However,intra-molecular links which make the adsorbed molecules even more rigid and less free to reorganize still occur.As a whole the results show the role of colloidal interactions and physical constraints in controlling the formation of enzyme-induced inter-molecular protein cross-linking.
Acknowledgments
Neil Rigby is greatly acknowledged for technical help and discussions on the results.This work was carried out in scope of the Marie Curie EU-project Enzymatic tailoring of protein interactions and functionalities in food matrix PRO-ENZ(MEST-CT-2005-020924) and was also supported by Academy of Finland Enzymatic cross-linking of food proteins:impact of food protein folding on the mode of action of cross-linking enzymes(No.:110965).Part of the experi-mental work was carried out during STSM granted by COST Action 928Control and exploitation of enzymes for added-value food products.
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D.Ercili-Cura et al./Food Hydrocolloids28(2012)1e99
免疫球蛋白的试题及答案
第四章免疫球蛋白 名词解释： 1．抗体(antibody) 2．Fab(fragment antigen binding) 3．Fc(fragment crytallizable) 4．免疫球蛋白(Immunoglobulin Ig) 5．超变区(hypervariable region，HVR) 6．可变区(variable region，V区) 7．单克隆抗体（Monoclonal antibody，mAb） 8．ADCC(Antibody –dependent cell-mediatedcytotoxicity) 9．调理作用(opsonization) 10．J链（joining chain） 11．分泌片（secretory piece） 12．Ig功能区（Ig domain） 13．Ig折叠（Ig folding） 14．CDR(complementary-determining region) 问答题 1．简述抗体与免疫球蛋白的区别和联系。 2．试述免疫球蛋白的主要生物学功能。 3．简述免疫球蛋白的结构、功能区及其功能。 4．简述单克隆抗体技术的基本原理。 参考答案 名词解释 1．抗体(Antibody) ：是B 细胞特异性识别Ag后，增殖分化成为浆细胞，所合成分泌的一类能与相应抗原特异性结合的、具有免疫功能的球蛋白。 2．Fab(Fragment antigen binding)：即抗原结合片段，每个Fab段由一条完整的轻链和重链的VH和CH1功能区构成，可以与抗原表位发生特异性结合。 3．Fc片段(fragment crytallizable)：即可结晶片段，相当于IgG的CH2和CH3功能区，无抗原结合活性，是抗体分子与效应分子和细胞相互作用的部位。 4. 免疫球蛋白(Immunoglobulin，Ig)：是指具有抗体活性或化学结构与抗体相似的球蛋白。可分为分泌型和膜型两类。 5．高变区（hypervariable region ，HVR）：在Ig分子VL和VH内，某些区域的氨基酸组成、排列顺序与构型更易变化，这些区域为超变区。 6．可变区（V区）：在Ig多肽链氨基端(N端)，L链1/2与H链1/4区域内，氨基酸的种类、排列顺序与构型变化很大，故称为可变区。 7．单克隆抗体(Monoclonal antibody ，mAb)：是由识别一个抗原决定簇的B淋巴细胞杂交瘤分裂而成的单一克隆细胞所产生的高度均一、高度专一性的抗体。 8．ADCC(Antibody –dependent cell-mediatedcytotoxicity)：即抗体依赖的细胞介导的细胞毒作用。是指表达Fc受体细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。NK细胞是介导ADCC的主要细胞。 9．调理作用（Opsonization）：是指IgG抗体（特别是IgG1和IgG3）的Fc段与中性粒细胞、巨噬细胞上的IgG Fc受体结合，从而增强吞噬细胞的吞噬作用。 10．J链（joining chain）：是由浆细胞合成的富含半胱氨酸的一条多肽链。J链可以连接Ig单体形成二聚体、五聚体或多聚体。
抗体的基本结构(精制甲类)
免疫球蛋白目录 1. 拼音 2. 英文参考 3. 概述 4. 免疫球蛋白分子的基本结构 1. 轻链和重链 2. 可变区和恒定区 3. 功能区 4. J链和分泌成分 5. 单体、双体和五聚体 6. 酶解片段 5. 免疫球蛋白分子的功能 1. 特异性结合抗原 2. 活化补体 3. 结合Fc受体 4. 通过胎盘 6. 免疫球蛋白分子的抗原性 1. 同种型 2. 同种异型 3. 独特型 7. 免疫球蛋白分子的超家族 1. 免疫球蛋白超家族的组成 2. 免疫球蛋白超家族的特点 8. 各类免疫球蛋白的生物学活性 1. IgG 2. IgA 3. IgM 4. IgD 5. IgE 9. 免疫球蛋白基因的结构和抗体多样性 1. Ig重链基因的结构和重排 2. Ig轻链基因的结构和重排 3. 抗体多样性的遗传学基础
10. 药理作用 11. 药品说明书 1. 适应症 2. 用量用法 12. 相关文献 具有抗体活性的血清蛋白称为免疫球蛋白，又称为抗体。是由机体的B淋巴细胞在抗原的刺激下分化、分裂而成的一组特殊球蛋白。人和动物的免疫血清中的免疫球蛋白极不均一，其组成、结构、大小、电荷、生物学活性等都有很大差异，约占机体全部血清蛋白的20～25％。目前已在人、小鼠等血清中先后分纯得到5类免疫球蛋白，1968年，世界卫生组织统一命名为免疫球蛋白G（IgG）、免疫球蛋白M（IgM）、免疫球蛋白A（IgA）、免疫球蛋白D（IgD）、免疫球蛋白E（IgE）。 免疫球蛋白分子的基本结构 Porter等对血清IgG抗体的研究证明，Ig单体分子的基本结构是由四条肽链组成的。即由二条相同的分子量较小的肽链称为轻链和二条相同的分子量较大的肽链称为重链组成的。轻链与重链是由二硫键连接形成一个四肽链分子称为Ig分子的单体，是构成免疫球蛋白分子的基本结构。Ig单体中四条肽链两端游离的氨基或羧基的方向是一致的，分别命名为氨基端（N端）和羧基端（C端）。 图2-3 免疫球蛋白分子的基本结构示意图 轻链和重链
抗原、抗体基本概念
一、抗原、抗体的概念及抗原抗体的关系 （一）抗原（Antigen） 凡能刺激机体产生抗体，并能与抗体发生特异性结合的物质称为抗原。物质所具有的这种特性称为抗原性（Antigenicity）。 （二）抗体 是机体受抗原刺激后，在体液中出现的一种能与相应抗原发生反应的球蛋白，称免疫球蛋白（Immunoglobulin, Ig）。含有免疫球蛋白的血清称免疫血清。 （三）抗原与抗体的关系 抗原是引起机体产生免疫反应的主要外因，决定免疫反应的特异性，机体与抗原物质的斗争过程中为加速循环和排除抗原而产生的抗体、致敏淋巴细胞等物质，是机体排除异体物质的保护性反应。没有抗原的刺激，机体不能产生抗体；没有抗原物质，也无法检测抗体的存在；利用抗体可以检测抗原物质。 二、抗原的性质及种类 （一）抗原的性质 1．异种异体物质机体能对进入体内的异种、异体的大分子物质产生抗体，该物质与机体的种类关系愈远，其抗原性就愈强，机体的免疫反应也更强。例如鸭血清蛋白对鸡的免疫原性较弱，而对家兔则能引起较强的免疫反应。 同种异体物质也可具有抗原性，同种不同个体之间，同一类型的细胞和组织，其抗原性也有差异，例如人的红细胞有ABO血型抗原及Rh型抗原。人类白细胞和其它组织的细胞膜上也具有组织相容性复合物的抗原物质（Man Histocompatibility complex, MHC）。 自身抗原：机体对本身所具有的物质不产生免疫反应。但在某些条件下，使机体某种物质、细胞或组织成分具有抗原性时，也可导致机体产生免疫反应。此具有抗原性的自身物质称自身抗原（Autoantigen），所产生的抗体称为自身抗体（Autoantibody）。如自身组织变性，机体组织或细胞在各种理化因素作用下，引起化学组成的分子排列和构型改变，形成新的抗原决定簇，例如服用安替比林、匹拉米洞等药所致白细胞减少，就是由于所服用药物改变了白细胞的一部分表面化学结构，形成新的抗原决定簇，激活免疫活性细胞产生白细胞抗体（自身抗体），导致白细胞减少症。在外伤、感染和炎症时，可能使隐蔽性抗原如精子、甲状腺球蛋白等释放，引起机体产生免疫反应。 并非异物都是抗原，例如砂尘和一些非生物性高分子聚合物，仅能激发细胞吞噬反应而不能使机体产生抗体或致敏淋巴细胞。 2．大分子胶体凡具有抗原性的物质，分子愈大，抗原性愈强（如细菌、蛋白质）。一般认为抗原分子量愈大，其表面积相应较大，接触免疫细胞机会增多，在体内停留时间较长，不易排除，因而对机体刺激作用也强。一般具有免疫原性的物质，其分子量常在10000以上。对于蛋白质组成的抗原，其分子量小于5000~10000免疫原性很弱或完全没有。但某些低分子量多肽、如胰岛素（分子量5734），升血糖激素（分子量3800），血管紧张素（分子量1031），对某些实验动物还是具有一定的免疫原性。分子量小的物质团聚成的多聚体或吸附于其它胶体（载体）表面，形成大分子表面结构时，如和蛋白质结合，即具有大分子胶体特性，可使小分子物质获得或增强抗原性，如细菌的多糖成分、青霉素等化学药物。 3．抗原的特异性各种抗原物质的化学组成虽然很复杂，但能刺激机体产生抗体并与抗体反应相结合的化学组成，仅仅是抗原物质表面的一些具有活性的化学基因－化学结构及空间构型，称为抗原物质决定簇（基）（Antigenic determinant）。各种抗原物质各有其特异的抗原决定簇，但不同的抗原物质常含有共同的抗原成分，称为类属抗原。在分类上相近的种类之间的同一类蛋白质抗原，可表现出类属抗原关系。多种物质结构的相似性，决定这些物质抗原上的类属关系，而分子结构的差异性，决定各种物质的抗原特异性。 抗原的特异性是临床诊断、预防、治疗的基础。各种特异诊断抗体的制备依靠特异性抗原物质的获得；在不易获得特异性抗原的条件下，可利用类属抗原代替。但在鉴别抗原时，应注意区分类属抗原，以免误诊。 一般认为，环状构型要比直线排列的分子免疫原性强，聚合状态的比单体强。具有大分子量的异物，无论具有何种构型，基本上具有免疫原性。但明胶和核酸免疫原性很弱或无。 免疫原的抗原决定簇是否暴露，抗原决定簇之间的距离是否适当，对于免疫原性强弱亦有很大影响。凡暴露的抗原决定簇的数目多，间距大，免疫原性也就较强。能与抗体分子结合的抗原决定簇的总数，称为抗原的结合价。简单的半抗原一般只能与一个抗体分子结合，是单价抗原。根据抗原分子大小推算，有100个氨基酸的多肽，约有14～20个不重叠的抗原决定簇，即有14～20个抗原结合价。 （二）抗原的种类
第四章 免疫球蛋白剖析
第四章免疫球蛋白 第一节基本概念 1、抗体：B淋巴细胞在有效的抗原刺激下分化为浆细胞，产生具有与相应抗原发生特异性结合功能的免疫球蛋白，这类免疫球蛋白称为抗体。 1937年，Tiselius用电泳方法将血清蛋白分为白蛋白、α1、α2、β及γ球蛋白等组分，其后又证明抗体的活性部分是在γ球蛋白部分。因此，相当长一段时间内，抗体又被称为γ球蛋白（丙种球蛋白）。实际上，抗体的活性除γ球蛋白外，还存在于α和β球蛋白处。 20世纪40年代初期，Tiselius和Kabat用肺炎球菌多糖免疫家兔，证实了抗体活性与血清丙种球蛋白组分相关。肺炎球菌多糖免疫家兔后可获得高效价免疫血清。然后加入相应抗原吸收以除去抗体，将除去抗体的血清进行电泳图谱分析，发现丙种球蛋白（γ-G）组分明显减少，从而证明了抗体活性是存在于丙种球蛋白内。 2、免疫球蛋白：具有抗体活性或化学结构与抗体相似的球蛋白统称为免疫球蛋白(immunoglobulin，Ig)。 区别： 抗体都是免疫球蛋白，而免疫球蛋白并不都是抗体。如骨髓瘤蛋白，巨球蛋白血症、冷球蛋白血症等患者血清中存在的异常免疫球蛋白结构与抗体相似，但无抗体活性。 免疫球蛋白可分为分泌型（secreted Ig,SIg）和膜型（membrane Ig, mIg）。 前者主要存在于血清及其他体液或外分泌液中，具有抗体的各种功能；后 者是B细胞表面的抗原识别受体。 第二节免疫球蛋白结构
一、免疫球蛋白的基本结构 （一）重链和轻链 免疫球蛋白分子是由两条相同的重链（heavy chain，H链）和两条相同的轻链（light chain，L链）通过链间二硫键连接而成的四肽链结构。X 射线晶体结构分析发现，IgG分子由3个相同大小的节段组成。 1. 重链 分子量约为50～75kD，由450～550个氨基酸残基组成。免疫球蛋白重链恒定区由于氨基酸的组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。不同的同种型具有不同的特征，包括链内二硫键的数目和位置、连接寡糖的数量、功能区的数目以及铰链区的长度等。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类。如IgG可分为IgG1～IgG4；IgA可分为IgA1和IgA2。IgM、IgD和IgE尚未发现有亚类。 2.轻链 免疫球蛋白轻链的分子量约25 kD，由214个氨基酸残基构成。轻链可分为两型，即κ(kappa)型和λ(lambda)型，一个天然Ig分子上两条轻链的型别总是相同的,两型轻链的功能无差异。不同种属中，两型轻链的比例不同，正常人血清免疫球蛋白κ:λ约为2:1，而在小鼠则为20:1。κ:λ比例的异常可能反映免疫系统的异常，例如人类免疫球蛋白λ链过多，提示可能有产生λ链的B细胞肿瘤。根据λ链恒定区个别氨基酸的差异，又可分为λ1、λ2、λ3和λ 4 四个亚型。 （二）可变区和恒定区 通过分析不同免疫球蛋白重链和轻链的氨基酸序列，发现重链和轻链靠近N端的约110个氨基酸的序列变化很大，称为可变区（variable
免疫球蛋白的结构
第一节免疫球蛋白的结构（The Structure of Immunoglobulin) B淋巴细胞在抗原刺激下增殖分化为浆细胞，产生能与相应抗原发生特异性结合的免疫蛋白，这类免疫球蛋白被称为抗体（antibody, Ab）。 1937年，Tiselius用电泳方法将血清蛋白分为白蛋白、α1、α2、β及γ球蛋白等组分，其后又证明抗体的活性部分是在γ球蛋白部分。因此，相当长一段时间内，抗体又被称为γ球蛋白（丙种球蛋白）。 实际上，抗体的活性除γ球蛋白外，还存在于α和β球蛋白处。1968年和1972年的两次国际会议上，将具有抗体活性或化学结构与抗体相似的球蛋白统一命名为免疫球蛋白（immunoglobulin，Ig）。 Ig是化学结构的概念，它包括正常的抗体球蛋白和一些未证实抗体活性的免疫球蛋白，如骨髓瘤病人血清中的M蛋白及尿中的本周氏（Bence Jones, BJ）蛋白等。 免疫球蛋白可分为分泌型（secreted Ig,SIg）和膜型（membrane Ig, mIg）。前者主要存在于血清及其他体液或外分泌液中，具有抗体的各种功能；后者是B细胞表面的抗原识别受体。 ☆☆相关素材☆☆ 图片正常人血清电泳分离图 一免疫球蛋白的基本结构 The basical structure of immunoglobulin 免疫球蛋白分子是由两条相同的重链（heavy chain，H链）和两条相同的轻链（light chain，L链）通过链间二硫键连接而成的四肽链结构。 X射线晶体结构分析发现，IgG分子由3个相同大小的节段组成，位于上端的两个臂由易弯曲的铰链区（hinge region）连接到主干上形成一个"Y"形分子，称为Ig分子的单体，是构成免疫球蛋白分子的基本单位。
抗体的基本结构
免疫球蛋白
具有抗体活性的血清蛋白称为免疫球蛋白，又称为抗体。是由机体的B淋巴细胞在抗原的刺激下分化、分裂而成的一组特殊球蛋白。人和动物的免疫血清中的免疫球蛋白极不均一，其组成、结构、大小、电荷、生物学活性等都有很大差异，约占机体全部血清蛋白的20～25％。目前已在人、小鼠等血清中先后分纯得到5类免疫球蛋白，1968年，世界卫生组织统一命名为免疫球蛋白G（IgG）、免疫球蛋白M（IgM）、免疫球蛋白A（IgA）、免疫球蛋白D（IgD）、免疫球蛋白E（IgE）。 Porter等对血清IgG抗体的研究证明，Ig单体分子的基本结构是由四条肽链组成的。即由二条相同的分子量较小的肽链称为轻链和二条相同的分子量较大的肽链称为重链组成的。轻链与重链是由二硫键连接形成一个四肽链分子称为Ig分子的单体，是构成免疫球蛋白分子的基本结构。Ig单体中四条肽链两端游离的氨基或羧基的方向是一致的，分别命名为氨基端（N端）和羧基端（C端）。 图2-3 免疫球蛋白分子的基本结构示意图 轻链和重链
由于骨髓瘤蛋白（M蛋白）是均一性球蛋白分子，并证明本周蛋白（BJ）是Ig分子的L链，很容易从患者血液和尿液中分离纯化这种蛋白，并可对来自不同患者的标本进行比较分析，从而为Ig分子氨基酸序列分析提供了良好的材料。 1．轻链（lightchain,L）轻链大约由214个氨基酸残基组成，通常不含碳水化合物，分子量约为24kD。每条轻链含有两个链内二硫键所组成的环肽。L链共有两型：kappa(κ)与lambda(λ)，同一个天然Ig分子上L链的型总是相同的。正常人血清中的κ：λ约为2：1。 2．重链（heavychain,H链）重链大小约为轻链的2倍，含450～550个氨基酸残基，分子量约为55或75kD。每条H链含有4～5个链内二硫键所组成的环肽。不同的H链由于氨基酸的排列顺序、二硫键的数目和们置、含糖的种类和数量不同，其抗原性也不相同，根据H链抗原性的差异可将其分为5类：μ链、γ链、α链、δ链和ε链，不同H链与L链（κ或λ链）组成完整Ig的分子分别称之为IgM、IgG、IgA、IgD和IgE。γ、α和δ链上含有4个环肽，μ和ε链含有5个环肽。重链（heavy chain,H链）由450～570个氨基酸残基组成，分子量约为50～70kD。不同的H链因氨基酸的排列顺序、二硫键的数目和位置、含糖的种类和数量不同，其抗原性也不相同，可将其分为μ链、γ链、α链、δ链、ε链五类，这些H链与L链（κ链或λ链）组成的完整Ig分子分别称为IgM（μ）、IgG（γ）、IgA （α）、IgD（δ）和IgE（ε 可变区和恒定区 通过对不同骨髓蛋白或本周蛋白H链或L链的氨基酸序列比较分析，发现其氨基端（N-末端）氨基酸序列变化很大，称此区为可变区（V），而羧基末端（C-末端）则相对稳定，变化很小，称此区为恒定区（C区）。 1．可变区（variableregion,V区）位于L链靠近N端的1/2（约含108～111个氨基酸残基）和H链靠近N端的1/5或1/4（约含118个氨基酸残基）。每个V区中均有一个由链内二硫键连接形成的肽环，每个肽环约含67～75个氨基酸残基。V区氨基酸的组成和排列随抗体结合抗原的特异性不同有较大的变异。由于V区中氨基酸的种类、排列顺序千变万化，故可形成许多种具有不同结合抗原特异性的抗体。 L链和H链的V区分别称为VL和VH。在VL和VH中某些局部区域的氨基酸组成和排列顺序具有更高的变休程度，这些区域称为高变区（hypervariable region,HVR）。在V 区中非HVR部位的氨基酸组面和排列相对比较保守，称为骨架区（framework region）。VL中的高变区有三个，通常分别位于第24～34、50～65、95～102位氨基酸。VL和VH 的这三个HVR分别称为HVR1、HVR2和HVR3。经X线结晶衍射的研究分析证明，高变区确实为抗体与抗原结合的位置，因而称为决定簇互补区（complementarity-determining region,CDR）。VL和VH的HVR1、HVR2和HVR3又可分别称为CDR1、CDR2和CDR3，
抗体的结构与功能
免疫球蛋白的结构与功能 一、免疫球蛋白分子的基本结构 Porter等对血清IgG抗体的研究证明，Ig分子的基本结构是由四肽链组成的。即由二条相同的分子量较小的肽链称为轻链和二条相同的分子量较大的肽链称为重链组成的。轻链与重链是由二硫键连接形成一个四肽链分子称为Ig分子的单体，是构成免疫球蛋白分子的基本结构。Ig单体中四条肽链两端游离的氨基或羧基的方向是一致的，分别命名为氨基端（N 端）和羧基端（C端）。 （一）轻链和重链 由于骨髓瘤蛋白（M蛋白）是均一性球蛋白分子，并证明本周蛋白（BJ）是Ig分子的L链，很容易从患者血液和尿液中分离纯化这种蛋白，并可对来自不同患者的标本进行比较分析，从而为Ig分子氨基酸序列分析提供了良好的材料。 1．轻链（light chain,L）轻链大约由214个氨基酸残基组成，通常不含碳水化合物，分子量约为24kD。每条轻链含有两个由链内二硫键内二硫所组成的环肽。L链共有两型：kappa(κ)与lambda(λ)，同一个天然Ig分子上L链的型总是相同的。正常人血清中的κ：λ约为2：1。 2．重链（heavy chain,H链）重链大小约为轻链的2倍，含450～550个氨基酸残基，分子量约为55或75kD。每条H链含有4～5个链内二硫键所组成的环肽。不同的H链由于氨基酸组成的排列顺序、二硫键的数目和们置、含的种类和数量不同，其抗原性也不相同，根据H链抗原性的差异可将其分为5类：μ链、γ链、α链、δ链和ε链，不同H链与L 链（κ或λ链）组成完整Ig的分子分别称之为IgM、IgG、IgA、IgD和IgE。γ、α和δ链上含有4个肽，μ和ε链含有5个环肽。 （二）可变区和恒定区 通过对不同骨髓蛋白或本周蛋白H链或L链的氨基酸序列比较分析，发现其氨基端（N-末端）氨基酸序列变化很大，称此区为可变区（V），而羧基末端（C-末端）则相对稳定，变化很小，称此区为恒定区。 1．可变区（variable region,V区）位于L链靠近N端的1/2（约含108～111个氨基酸残基）和H链靠近N端的1/5或1/4（约含118个氨基酸残基）。每个V区中均有一个由链内二硫键连接形成的肽环，每个肽环约含67～75个氨基酸残基。V区氨基酸的组成和排列随抗体结合抗原的特异性不同有较大的变异。由于V区中氨基酸的种类为排列顺序千变万化，故可形成许多种具有不同结合抗原特异性的抗体。 L链和H链的V区分别称为VL和VH。在VL和VH中某些局部区域的氨基酸组成和排列顺序具有更高的变休程度，这些区域称为高变区（hypervariable region,HVR）。在V区中非HVR部位的氨基酸组面和排列相对比较保守，称为骨架区（fuamework rugion）。VL 中的高变区有三个，通常分别位于第24～34、50～65、95～102位氨基酸。VL和VH的这三个HVR分别称为HVR1、HVR2和HVR3。经X线结晶衍射的研究分析证明，高变区确实为抗体与抗原结合的位置，因而称为决定簇互补区（complementarity-determining regi-on,CDR）。VL和VH的HVR1、HVR2和HVR3又可分别称为CDR1、CDR2和CDR3，一般的CDR3具有更高的高变程度。高变区也是Ig分子独特型决定簇（idiotypic determinants）主要存在的部位。在大多数情况下H链在与抗原结合中起更重要的作用。 2．恒定区（constant region,C区）位于L链靠近C端的1/2（约含105个氨基酸残基）和H 链靠近C端的3/4区域或4/5区域（约从119位氨基酸至C末端）。H链每个功能区约含110多个氨基酸残基，含有一个由二锍键连接的50～60个氨基酸残基组成的肽环。这个区域氨
免疫球蛋白的结构
第一节免疫球蛋白的结构 (The Structure of Immunoglobulin) B淋巴细胞在抗原刺激下增殖分化为浆细胞，产生能与相应抗原发生特异性结合的免疫蛋 白，这类免疫球蛋白被称为抗体( an tibody, Ab )。 1937年，Tiselius 用电泳方法将血清蛋白分为白蛋白、a 1、a 2、B及丫球蛋白等组分，其后又证明抗体的活性部分是在丫球蛋白部分。因此，相当长一段时间内，抗体又被称为丫 球蛋白(丙种球蛋白)。 实际上，抗体的活性除丫球蛋白外，还存在于a和B球蛋白处。1968年和1972年的两次 国际会议上，将具有抗体活性或化学结构与抗体相似的球蛋白统一命名为免疫球蛋白(immunoglobulin , Ig )。 Ig是化学结构的概念，它包括正常的抗体球蛋白和一些未证实抗体活性的免疫球蛋白，如骨髓瘤病人血清中的M蛋白及尿中的本周氏(Be nee Jon es, BJ )蛋白等。 免疫球蛋白可分为分泌型(secreted lg,Slg )和膜型(membrane Ig, mIg )。前者主要存在于血清及其他体液或外分泌液中，具有抗体的各种功能；后者是B细胞表面的抗原识别 受体。 ☆☆相关素材☆☆ 图片正常人血清电泳分离图 I 丨总血清 -------- igG -------- IgA --------- IgM 一电泳迁移率十 (igES极少、不能定曲表示) 正常人血清电泳分离图 一免疫球蛋白的基本结构The basical structure of immunoglobulin 免疫球蛋白分子是由两条相同的重链( heavy chain , H链)和两条相同的轻链(light chain , L链)通过链间二硫键连接而成的四肽链结构。 X射线晶体结构分析发现，IgG分子由3个相同大小的节段组成，位于上端的两个臂由易弯曲的铰链区(hinge region )连接到主干上形成一个 "Y"形分子，称为Ig分子的单体, 是构成免疫球蛋白分子的基本单位。
抗体的基本结构
1.适应症 2.用量用法 12.相关文献 具有抗体活性得血清蛋白称为免疫球蛋白，又称为抗体。就是由机体得B淋巴细胞在抗原得刺激下分化、分裂而成得一组特殊球蛋白。人与动物得免疫血清中得免疫球蛋白极不均一，其组成、结构、大小、电荷、生物学活性等都有很大差异，约占机体全部血清蛋白得20～25％。目前已在人、小鼠等血清中先后分纯得到5类免疫球蛋白，1968年，世界卫生组织统一命名为免疫球蛋白G（IgG）、免疫球蛋白M（IgM）、免疫球蛋白A（IgA）、免疫球蛋白D（IgD）、免疫球蛋白E（IgE）。 免疫球蛋白分子得基本结构 Porter等对血清IgG抗体得研究证明，Ig单体分子得基本结构就是由四条肽链组成得。即由二条相同得分子量较小得肽链称为轻链与二条相同得分子量较大得肽链称为重链组成得。轻链与重链就是由二硫键连接形成一个四肽链分子称为Ig分子得单体，就是构成免疫球蛋白分子得基本结构。Ig单体中四条肽链两端游离得氨基或羧基得方向就是一致得，分别命名为氨基端（N端）与羧基端（C端）。 图2-3 免疫球蛋白分子得基本结构示意图 轻链与重链 由于骨髓瘤蛋白（M蛋白）就是均一性球蛋白分子，并证明本周蛋白（BJ）就是Ig分子得L链，很容易从患者血液与尿液中分离纯化这种蛋白，并可对来自不同患者得标本进行比较分析，从而为Ig分子氨基酸序列分析提供了良好得材料。
1．轻链（lightchain,L）轻链大约由214个氨基酸残基组成，通常不含碳水化合物，分子量约为24kD。每条轻链含有两个链内二硫键所组成得环肽。L链共有两型：kappa(κ)与lambda(λ)，同一个天然Ig分子上L链得型总就是相同得。正常人血清中得κ：λ约为2：1。 2．重链（heavychain,H链）重链大小约为轻链得2倍，含450～550个氨基酸残基，分子量约为55或75kD。每条H链含有4～5个链内二硫键所组成得环肽。不同得H链由于氨基酸得排列顺序、二硫键得数目与们置、含糖得种类与数量不同，其抗原性也不相同，根据H链抗原性得差异可将其分为5类：μ链、γ链、α链、δ链与ε链，不同H链与L链（κ或λ链）组成完整Ig得分子分别称之为IgM、IgG、IgA、IgD与IgE。γ、α与δ链上含有4个环肽，μ与ε链含有5个环肽。重链（heavy chain,H链）由450～570个氨基酸残基组成，分子量约为50～70kD。不同得H链因氨基酸得排列顺序、二硫键得数目与位置、含糖得种类与数量不同，其抗原性也不相同，可将其分为μ链、γ链、α链、δ链、ε链五类，这些H链与L链（κ链或λ链）组成得完整Ig分子分别称为IgM（μ）、IgG（γ）、IgA（α）、IgD（δ）与IgE（ε 可变区与恒定区 通过对不同骨髓蛋白或本周蛋白H链或L链得氨基酸序列比较分析，发现其氨基端（N-末端）氨基酸序列变化很大，称此区为可变区（V），而羧基末端（C-末端）则相对稳定，变化很小，称此区为恒定区（C区）。 1．可变区（variableregion,V区）位于L链靠近N端得1/2（约含108～111个氨基酸残基）与H链靠近N端得1/5或1/4（约含118个氨基酸残基）。每个V区中均有一个由链内二硫键连接形成得肽环，每个肽环约含67～75个氨基酸残基。V区氨基酸得组成与排列随抗体结合抗原得特异性不同有较大得变异。由于V区中氨基酸得种类、排列顺序千变万化，故可形成许多种具有不同结合抗原特异性得抗体。 L链与H链得V区分别称为VL与VH。在VL与VH中某些局部区域得氨基酸组成与排列顺序具有更高得变休程度，这些区域称为高变区（hypervariable region,HVR）。在V区中非HVR部位得氨基酸组面与排列相对比较保守，称为骨架区（framework region）。VL中得高变区有三个，通常分别位于第24～34、50～65、95～102位氨基酸。VL与VH得这三个HVR分别称为HVR1、HVR2与HVR3。经X线结晶衍射得研究分析证明，高变区确实为抗体与抗原结合得位置，因而称为决定簇互补区（plementarity-determining region,CDR）。VL 与VH得HVR1、HVR2与HVR3又可分别称为CDR1、CDR2与CDR3，一般得CDR3具有更高得高变程度。高变区也就是Ig分子独特型决定簇（idiotypic determinants）主要存在得部位。在大多数情况下H链在与抗原结合中起更重要得作用。
抗体的结构与功能
来源：医学全在线更新：2007-12-3 医学论坛 该文章转载自医学全在线：https://www.sodocs.net/doc/945904575.html,/edu/200712/18959.shtml 免疫球蛋白的结构与功能 一、免疫球蛋白分子的基本结构 Porter等对血清IgG抗体的研究证明，Ig分子的基本结构是由四肽链组成的。即由二条相同的分子量较小的肽链称为轻链和二条相同的分子量较大的肽链称为重链组成的。轻链与重链是由二硫键连接形成一个四肽链分子称为Ig分子的单体，是构成免疫球蛋白分子的基本结构。Ig单体中四条肽链两端游离的氨基或羧基的方向是一致的，分别命名为氨基端（N 端）和羧基端（C端）。 图2-3 免疫球蛋白分子的基本结构示意图 （一）轻链和重链 由于骨髓瘤蛋白（M蛋白）是均一性球蛋白分子，并证明本周蛋白（BJ）是Ig分子的L链，很容易从患者血液和尿液中分离纯化这种蛋白，并可对来自不同患者的标本进行比较分析，从而为Ig分子氨基酸序列分析提供了良好的材料。 1．轻链（light chain,L）轻链大约由214个氨基酸残基组成，通常不含碳水化合物，分子量约为24kD。每条轻链含有两个由链内二硫键内二硫所组成的环肽。L链共有两型：
kappa(κ)与lambda(λ)，同一个天然Ig分子上L链的型总是相同的。正常人血清中的κ：λ约为2：1。 2．重链（heavy chain,H链）重链大小约为轻链的2倍，含450～550个氨基酸残基，分子量约为55或75kD。每条H链含有4～5个链内二硫键所组成的环肽。不同的H链由于氨基酸组成的排列顺序、二硫键的数目和们置、含的种类和数量不同，其抗原性也不相同，根据H链抗原性的差异可将其分为5类：μ链、γ链、α链、δ链和ε链，不同H链与L 链（κ或λ链）组成完整Ig的分子分别称之为IgM、IgG、IgA、IgD和IgE。γ、α和δ链上含有4个肽，μ和ε链含有5个环肽。 （二）可变区和恒定区 通过对不同骨髓蛋白或本周蛋白H链或L链的氨基酸序列比较分析，发现其氨基端（N-末端）氨基酸序列变化很大，称此区为可变区（V），而羧基末端（C-末端）则相对稳定，变化很小，称此区为恒定区。 1．可变区（variable region,V区）位于L链靠近N端的1/2（约含108～111个氨基酸残基）和H链靠近N端的1/5或1/4（约含118个氨基酸残基）。每个V区中均有一个由链内二硫键连接形成的肽环，每个肽环约含67～75个氨基酸残基。V区氨基酸的组成和排列随抗体结合抗原的特异性不同有较大的变异。由于V区中氨基酸的种类为排列顺序千变万化，故可形成许多种具有不同结合抗原特异性的抗体。 L链和H链的V区分别称为VL和VH。在VL和VH中某些局部区域的氨基酸组成和排列顺序具有更高的变休程度，这些区域称为高变区（hypervariable region,HVR）。在V区中非HVR部位的氨基酸组面和排列相对比较保守，称为骨架区（fuamework rugion）。VL 中的高变区有三个，通常分别位于第24～34、50～65、95～102位氨基酸。VL和VH的这三个HVR分别称为HVR1、HVR2和HVR3。经X线结晶衍射的研究分析证明，高变区确实为抗体与抗原结合的位置，因而称为决定簇互补区（complementarity-determining regi-on,CDR）。VL和VH的HVR1、HVR2和HVR3又可分别称为CDR1、CDR2和CDR3，一般的CDR3具有更高的高变程度。高变区也是Ig分子独特型决定簇（idiotypic determinants）主要存在的部位。在大多数情况下H链在与抗原结合中起更重要的作用。
免疫球蛋白分子的结构与功能
、免疫球蛋白分子的基本结构 Porter等对血清IgG 抗体的研究证明，lg分子的基本结构是由四肽链组成的。即由二条 相同的分子量较小的肽链称为轻链和二条相同的分子量较大的肽链称为重链组成的。轻链与重链是由二硫键连接形成一个四肽链分子称为lg分子的单体，是构成免疫球蛋白分子的基 本结构。lg单体中四条肽链两端游离的氨基或羧基的方向是一致的，分别命名为氨基端（N 端）和羧基端（C端）。 图2-3免疫球蛋白分子的基本结构示意图 （一）轻链和重链 由于骨髓瘤蛋白（M蛋白）是均一性球蛋白分子，并证明本周蛋白（BJ）是lg分子的 L链，很容易从患者血液和尿液中分离纯化这种蛋白，并可对来自不同患者的标本进行比较 分析，从而为lg分子氨基酸序列分析提供了良好的材料。 1. 轻链（light chain,L ）轻链大约由214个氨基酸残基组成，通常不含碳水化合物，分子量约为24kD。每条轻链含有两个由链内二硫键内二硫所组成的环肽。L链共有两型：kappa（与lambda（入）同一个天然lg分子上L链的型总是相同的。正常人血清中的K入约为2：1。 2. 重链（heavy chain,H链）重链大小约为轻链的2倍，含450?550个氨基酸残基，分子量约为55或75kD。每条H链含有4?5个链内二硫键所组成的环肽。不同的H链由于 ?戰水化合韧
氨基酸组成的排列顺序、二硫键的数目和们置、含的种类和数量不同，其抗原性也不相同，根据H链抗原性的差异可将其分为5类：卩链、丫链、a链、3链和￡链，不同H链与L链 （K或入链）组成完整Ig的分子分别称之为IgM、IgG、IgA、IgD和IgE。Y a和3链上含有4个肽，□和&链含有5个环肽。 （二）可变区和恒定区 通过对不同骨髓蛋白或本周蛋白H链或L链的氨基酸序列比较分析，发现其氨基端（N- 末端）氨基酸序列变化很大，称此区为可变区（V），而羧基末端（C-末端）则相对稳定，变化很小，称此区为恒定区。 1. 可变区（variable region,V区）位于L链靠近N端的1/2 （约含108?111个氨基酸残基）和H链靠近N端的1/5或1/4 （约含118个氨基酸残基）。每个V 区中均有一个由链内二硫键连接形成的肽环，每个肽环约含67?75个氨基酸残基。V区氨基酸的组成和排列 随抗体结合抗原的特异性不同有较大的变异。由于V区中氨基酸的种类为排列顺序千变万 化，故可形成许多种具有不同结合抗原特异性的抗体。 L链和H链的V区分别称为VL和VH。在VL和VH中某些局部区域的氨基酸组成和排列顺序具有更高的变休程度，这些区域称为高变区（hypervariable region,HVR ）。在V区 中非HVR部位的氨基酸组面和排列相对比较保守，称为骨架区（fuamework rugion ）。VL 中的高变区有三个，通常分别位于第24?34、50?65、95?102位氨基酸。VL和VH的这 三个HVR分别称为HVR1、HVR2和HVR3。经X线结晶衍射的研究分析证明，高变区确实为抗体与抗原结合的位置，因而称为决定簇互补区（compleme ntarity-determi ning regi-on,CDR）o VL 和VH 的HVR1、HVR2 和HVR3 又可分另U称为CDR1、CDR2 和CDR3 , 一般的CDR3具有更高的高变程度。高变区也是Ig分子独特型决定簇（idiotypic determ inants 主要存在的部位。在大多数情况下H链在与抗原结合中起更重要的作用。
免疫球蛋白分子的结构与功能
一、免疫球蛋白分子的基本结构 Porter等对血清IgG抗体的研究证明，Ig分子的基本结构是由四肽链组成的。即由二条相同的分子量较小的肽链称为轻链和二条相同的分子量较大的肽链称为重链组成的。轻链与重链是由二硫键连接形成一个四肽链分子称为Ig分子的单体，是构成免疫球蛋白分子的基本结构。Ig单体中四条肽链两端游离的氨基或羧基的方向是一致的，分别命名为氨基端（N 端）和羧基端（C端）。 图2-3 免疫球蛋白分子的基本结构示意图 （一）轻链和重链 由于骨髓瘤蛋白（M蛋白）是均一性球蛋白分子，并证明本周蛋白（BJ）是Ig分子的L链，很容易从患者血液和尿液中分离纯化这种蛋白，并可对来自不同患者的标本进行比较分析，从而为Ig分子氨基酸序列分析提供了良好的材料。 1．轻链（light chain,L）轻链大约由214个氨基酸残基组成，通常不含碳水化合物，分子量约为24kD。每条轻链含有两个由链内二硫键内二硫所组成的环肽。L链共有两型：kappa(κ)与lambda(λ)，同一个天然Ig分子上L链的型总是相同的。正常人血清中的κ：λ约为2：1。 2．重链（heavy chain,H链）重链大小约为轻链的2倍，含450～550个氨基酸残基，分子量约为55或75kD。每条H链含有4～5个链内二硫键所组成的环肽。不同的H链由于
氨基酸组成的排列顺序、二硫键的数目和们置、含的种类和数量不同，其抗原性也不相同，根据H链抗原性的差异可将其分为5类：μ链、γ链、α链、δ链和ε链，不同H链与L链（κ或λ链）组成完整Ig的分子分别称之为IgM、IgG、IgA、IgD和IgE。γ、α和δ链上含有4个肽，μ和ε链含有5个环肽。 （二）可变区和恒定区 通过对不同骨髓蛋白或本周蛋白H链或L链的氨基酸序列比较分析，发现其氨基端（N-末端）氨基酸序列变化很大，称此区为可变区（V），而羧基末端（C-末端）则相对稳定，变化很小，称此区为恒定区。 1．可变区（variable region,V区）位于L链靠近N端的1/2（约含108～111个氨基酸残基）和H链靠近N端的1/5或1/4（约含118个氨基酸残基）。每个V区中均有一个由链内二硫键连接形成的肽环，每个肽环约含67～75个氨基酸残基。V区氨基酸的组成和排列随抗体结合抗原的特异性不同有较大的变异。由于V区中氨基酸的种类为排列顺序千变万化，故可形成许多种具有不同结合抗原特异性的抗体。 L链和H链的V区分别称为VL和VH。在VL和VH中某些局部区域的氨基酸组成和排列顺序具有更高的变休程度，这些区域称为高变区（hypervariable region,HVR）。在V区中非HVR部位的氨基酸组面和排列相对比较保守，称为骨架区（fuamework rugion）。VL 中的高变区有三个，通常分别位于第24～34、50～65、95～102位氨基酸。VL和VH的这三个HVR分别称为HVR1、HVR2和HVR3。经X线结晶衍射的研究分析证明，高变区确实为抗体与抗原结合的位置，因而称为决定簇互补区（complementarity-determining regi-on,CDR）。VL和VH的HVR1、HVR2和HVR3又可分别称为CDR1、CDR2和CDR3，一般的CDR3具有更高的高变程度。高变区也是Ig分子独特型决定簇（idiotypic determinants）主要存在的部位。在大多数情况下H链在与抗原结合中起更重要的作用。
抗体结构与分类
抗体结构与分类 大多数哺乳动物的抗体基本结构是一个由四条多肽链（二硫键连接的二条重链和二条轻链）组成的糖基化蛋白，分子量约150，000Da。轻链的分子量约25，000Da，由二个结构域组成，一个可变区V L 和一个恒定区C L。轻链有κ和λ两种类型，人的L 链中κ型占60％，λ型占40％；小鼠L 链中κ型和λ型分别为95％和5％。一个抗体分子中的L 链只有一种类型。 重链分子量约50，000Da，有恒定区和可变区组成。轻链和重链有很多相似氨基酸序列构成的同源区。这些同源区有110 个氨基酸，称为免疫球蛋白结构域。重链包括可变区V H 和3～4 个恒定区，C H1, C H2, C H3,和C H4（依抗体类型不同）。C H1 和C H2 之间有一个铰链区，使得Y 型抗体分子的两个Fab 臂具有灵活性，以结合固定距离的两个抗原决定簇。 重链也决定抗体分子的功能活性。依据重链不同，抗体分子分为IgG, IgA, IgM, IgE 和IgD，对应的重链分别为, , μ, 和。IgD, IgE, 和 IgG 通常以单体存在，IgA 有单体和二聚体两种形式，IgM 以五聚体存在，由二硫键连接。IgG 依产生物种不同又分为四个轻微差异的亚型，称为同型。
蛋白水解酶水解IgG 形成有特定生物特性的固定片段，有助于IgG 结构和功能的研究。胃蛋白酶作用于IgG 分子，产生F(ab')2 片段，包括铰链区连接的两个Fab 区。 F(ab')2 分子是二价的，可作用于抗原。 木瓜蛋白酶水解IgG 时作用在C H1 和C H2 之间的铰链区，产生两个单独的Fab 片段和一个Fc 片段。Fab 有抗原结合活性，Fc 则没有。Fc 是糖基化的片段，具有很多效应功能（如结合补体、结合巨噬细胞和单核细胞的细胞受体等），也可用于划分抗体类型。
抗体的基本结构
抗体的基本结构
免疫球蛋白
具有抗体活性的血清蛋白称为免疫球蛋白，又称为抗体。是由机体的B淋巴细胞在抗原的刺激下分化、分裂而成的一组特殊球蛋白。人和动物的免疫血清中的免疫球蛋白极不均一，其组成、结构、大小、电荷、生物学活性等都有很大差异，约占机体全部血清蛋白的20～25％。目前已在人、小鼠等血清中先后分纯得到5类免疫球蛋白，1968年，世界卫生组织统一命名为免疫球蛋白G
（IgG）、免疫球蛋白M（IgM）、免疫球蛋白A（IgA）、免疫球蛋白D （IgD）、免疫球蛋白E（IgE）。 免疫球蛋白分子的基本结构 Porter等对血清IgG抗体的研究证明，Ig单体分子的基本结构是由四条肽链组成的。即由二条相同的分子量较小的肽链称为轻链和二条相同的分子量较大的肽链称为重链组成的。轻链与重链是由二硫键连接形成一个四肽链分子称为Ig分子的单体，是构成免疫球蛋白分子的基本结构。Ig单体中四条肽链两端游离的氨基或羧基的方向是一致的，分别命名为氨基端（N端）和羧基端（C 端）。 图2-3 免疫球蛋白分子的基本结构示意图 轻链和重链 由于骨髓瘤蛋白（M蛋白）是均一性球蛋白分子，并证明本周蛋白（BJ）是Ig分子的L链，很容易从患者血液和尿液中分离纯化这种蛋白，并可对来自
不同患者的标本进行比较分析，从而为Ig分子氨基酸序列分析提供了良好的材料。 1．轻链（lightchain,L）轻链大约由214个氨基酸残基组成，通常不含碳水化合物，分子量约为24kD。每条轻链含有两个链内二硫键所组成的环肽。L 链共有两型：kappa(κ)与lambda(λ)，同一个天然Ig分子上L链的型总是相同的。正常人血清中的κ：λ约为2：1。 2．重链（heavychain,H链）重链大小约为轻链的2倍，含450～550个氨基酸残基，分子量约为55或75kD。每条H链含有4～5个链内二硫键所组成的环肽。不同的H链由于氨基酸的排列顺序、二硫键的数目和们置、含糖的种类和数量不同，其抗原性也不相同，根据H链抗原性的差异可将其分为5类：μ链、γ链、α链、δ链和ε链，不同H链与L链（κ或λ链）组成完整Ig 的分子分别称之为IgM、IgG、IgA、IgD和IgE。γ、α和δ链上含有4个环肽，μ和ε链含有5个环肽。重链（heavy chain,H链）由450～570个氨基酸残基组成，分子量约为50～70kD。不同的H链因氨基酸的排列顺序、二硫键的数目和位置、含糖的种类和数量不同，其抗原性也不相同，可将其分为μ链、γ链、α链、δ链、ε链五类，这些H链与L链（κ链或λ链）组成的完整Ig分子分别称为IgM（μ）、IgG（γ）、IgA（α）、IgD（δ）和IgE（ε 可变区和恒定区 通过对不同骨髓蛋白或本周蛋白H链或L链的氨基酸序列比较分析，发现其氨基端（N-末端）氨基酸序列变化很大，称此区为可变区（V），而羧基末端（C-末端）则相对稳定，变化很小，称此区为恒定区（C区）。