Vol. 261, No. 31, Issue of Novemhe!
THEJOURNAL OF BIOLOGICAL
CHEMISTRY
0 1986 hy The American Society of Biological Chemists, Inc
14621-14627.1986
Printed in U.S . A.
Isolation, Macromolecular Properties, andCombining Site of a Chitooligosaccharide-specific Lectin from the Exudateof Ridge Gourd
(Luffaacutangzda)*
(Received for publication, July 8, 1985)
Vellareddy Anantharam$, Sankhavaram R. Patanjalij, M. Joginadha Swamys,Ashok R. Sanadiq,
Irwin J. Goldsteinll, and Avadhesha Surolia**
From the Molecular Biophysics Unit, Indian Instituteof Science, Bangalore 560 012, India and the )IDepartment of Biological
Chemistry, The Universityof Michigan, Ann Arbor, Michigan48109
* This investigation was supported by a grant from the Department
of Science and Technology (India) and partially supported by Grant
FG-IN-662 made by the United States Department of Agriculture (to
A. S.) and National Institutes of Health Grant GM 29470 (to I. J.
G.). The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
$ Senior Research Fellow of the University Grants Commission,
India.
3 Senior Research Fellow in a project funded by Council of Scientific and Industrial Research, India.
ll Senior Research Fellow of the Department of Science and Technology grant funded to A. S.
** To whom all correspondence must be addressed.
tetra-N-acetylchitotetraose, N,N’,N”,N”-tetraaacetylchitotetraose; penta-N-acetylchitopentaose,N,N,N”,N“‘,N“”-pentaacetylchitapentaose; ManNac, N-acetyl-D-mannosamine; LacNAc, N-acetyllactosasmine; MeaGlcNAc, methyl-2-acetarnido-2-deoxy-a-~-glucopyranoside (MePGlcNAc refers to (3 anomer); PBS, phosphatebuffered saline (0.02 M sodium phosphate buffer (pH 7.4) containing
0.15 M NaCI); PME, P-mercaptoethanol; GuHCI, guanidine hydrochloride; SDS, sodium dodecyl sulfate; ConA, concanavalin A WGA,
wheat germ agglutinin; PAGE, polyacrylamide gel electrophoresis;
dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.
Appeared as abstracts in the following: Anantharam, V., Patanjali, S. R., and Surolia, A. (1983) in the Third Congress of the
Federation of Asian and Oceanian Biochemists, Bangkok, Thailand,
Nov. 29-Dec. 2, 1983, p. 75; Surolia, A. (1984) in the International
Symposium on Biomoleculer Structure and Interactions, Bangalore,
India, Dec. 17-22, 1984, p. 43.
14621
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A lectin specific for chito-oligosaccharides from the increasingly being employed as highly discriminating macroexudate of ridge gourd (Luffaacutangula) fruits has molecular probes in lymphocyte mitogenesis, in the purificabeen purified to homogeneity by affinity chromatog- tion of glycoproteins, in the study of cell surfaces of normal
raphy. The lectin has
a molecular weight of 48,000, an and cancerous cells, etc. (3, 4).
Lectins were initially discovered in plant seeds and thereSX,,,~ of 4.06 S and a Stokes radius of 2.9 nm. Upon
sodium dodecyl sulfate-polyacrylamide gel electropho- fore, these lectins have been most widely studied. Reports on
to of 24,000 was their role in plants which relate their turnover in seeds may
resis, a single band corresponding M,
observed both in the presence and absence of &mer- shed light on their functions indevelopment and differentiacaptoethanol. The subunits in this dimeric lectin are, tion, andin root-bacterium symbiosis. Lectins, however, have
therefore, held together solely by noncovalent inter- also been found in other parts of the plant (2, 9). Currently,
actions. The lectinis not a glycoprotein, and secondary major efforts are being directed toward elucidating the relastructure analysis by CD measurements showed 31% tionship of lectins isolated from various parts of the plants
a-helix. The hemagglutinating activity of L. acutan- and their overall function. In this regard, the discovery of a
gula agglutinin was not inhibited by any of the mono- lectin from the phloem exudate of pumpkin (Cucurbita m u saccharides tested. Among the disaccharides only di- h a ) and some other cucurbits (10) provides an opportunity
N-acetylchitobiose was inhibitory. The inhibitory po- to investigate their functions in view of their unusual location
tency of chito-oligosaccharides increased dramatically in plants. The sugar specificity of these lectins was shown to
with theirsize up to penta-N-acetylchltopentaose.The be directed toward di-N-acetylchitobiose’ ( l l ) , similar to the
lectin has two binding sites for saccharides. The affinlectins from wheat germ (12), potato tubers (13), thornapple
ity of chito-oligosaccharides for L. acutangula lectin, (14), and tomato fruits (15, 16). In this study we report the
as monitored by titrating thechanges in the nearUV- isolation and characterization of a chito-oligosaccharide-bindCD spectra and intrinsic
fluorescence, increased strik- ing lectin discovered in the exudate of ridgegourd (Luffu
ingly with the number of GlcNAc units in them. The acutangula) fruits as prelude
a
to our studies on the structurevalues of AG, AH, and A S €or the binding process function relationship of exudate lectins. Lectins binding to
showed a pronounced dependence on the size of the GlcNAc or its oligomers have been described in the recent
chito-oligosaccharides, indicating that the binding of years; however, they appear to exhibit multiple specificities
higher oligomers is progressively more favored ther- for binding to carbohydrate ligands (17-19), Therefore, the L.
modynamically than di-N-acetylchitobiose. The ther- acutangula lectin should prove to be a valuable probe for
modynamic data are consistent with an extended bind- studying the expression, biogenesis, and structureof chitobioing sitein this lectin, which accommodates a tetrasac- syl-containing glycoproteins. We show that this lectin poscharide.
sesses a rather highly extended binding site which accommodates a tetrasaccharide.This lectin, therefore, differs from all
other GlcNAc-binding lectins including those from the other
cucurbits? The most likely arrangement of the GlcNAc units
Lectins, a class of hemagglutinating proteins are ubiqui- in the combining region of this lectin is also proposed.
tously distributed in nature (1-5). Because of the high degree
of selectivity shown by the individual lectins for their interThe abbreviations used are: di-N-acetylchitobios, N,N’-diaceactions with glycoproteins and glycolipids (6-8), lectins are tylchitobiose; tri-N-acetylchitotriose, N,N‘,N“-triacetylchitotriose;
14622
A Tetra-N-acetylchitotetraose-specific Exudate Lectin
binding
Better
orientation
of the
di-N-acetylchitobiosyl sequence in the core region of these glycopeptides is,
DISCUSSION
therefore presumably responsible for the higher potency of
This report describes the purification and characterization glycopeptides whencompared tochito-oligosaccharides them.~
dialysis data show that the stoichiometry
of a highly specific, chito-oligosaccharide-binding lectin from ~ e l v e sEquilibrium
1 per polypeptide chain.
the exudate of L. acutangula fruits. A molecular weight of for the lectin-sugar interaction is
The
near
UV-CD
spectrum
of
L.
acutangula lectin shows
48,000 for the native protein and 24,000 for the denatured
protein obtained by several methods allowed us t o conclude considerableligand concentrationandsize-dependentenellipticities. An increasein A6 in the
that the protein iscomposed of two identical subunits. Since hancements in the molar
difference
spectrum
of
the
protein corresponding to the trypthe molecular weight of the native lectin as
well as thatof its
subunits obtained by denaturation with sodium dodecyl sul- tophanyl side chains correlates qualitatively with the molar
fateorguanidine HC1 did not vary on treatment with ,B- ellipticity changes observed in the near UV-CD spectrum,
mercaptoethanol, disulfide bonds are apparently notinvolved suggesting that most of these changes can be ascribed to
in subunit association of this dimeric, cysteine-containing perturbation of tryptophan absorbance in the protein?
fluorescence, which is typilectin. Its hydrodynamic properties are consistent with those The enhancement in the lectin
expected for a globular protein. L. acutangula lectin is char- cal of tryptophan emission in proteins, and its blue shift on
of
acterized by a high content of a-helix. The absence of ,B- binding t o chito-oligosaccharides are related to the number
GlcNAc units in these saccharides and reflect some unique
pleated structure appears to be
a distinct feature of this
spatial disposition of the bound saccharides with respect ato
protein when compared with most other lectins(70-72).
tryptophan residue in the binding site.7 Increments in blue
Unlike wheat germ agglutinin (WGA) (73), the agglutinatshift indicate a progressive increase in hydrophobicity of the
by GlcNAc
ing activityof L. acutangula lectin is not inhibited
environment around the tryptophan residue(s)with increase
or its methylglycosides. In this respect itdiffers from potato in thesize of the saccharide.
(72), thorn apple (17), and other cucurbits(11)as well, which
The association constants(K,) obtained by fluorescence as
are weakly inhibited by GlcNAc. TheotherN-acetylated
well as near UV-CD titrations differ greatly from one ligand
hexosamines tested were also ineffective. This lectin is spe- to another. The binding affinitiesof L. acutangula lectin for
cifically inhibited by di-N-acetylchitobiose and higheroligo- various saccharides as obtained from fluorescence and CD
mers of GlcNAc, and their potency increases strikinglywith spectroscopy show a more pronounced effect on ligand size
increase in their size. When compared with WGA, which is than that observed for potato lectin and WGA (72, 79). The
almost equally inhibited by tri-N-acetylchitotriose and tetra- affinity of penta-N-acetylchitopentaose,tetra-N-acetylchitoN-acetylchitotetraose, and the Cucurbita pepo lectin where tetraose, and tri-N-acetylchitotriose is about 550, 78, and 9
di-, tri-, and tetrasaccharides are
equally potent inhibitors
times greater, respectively, than di-N-acetylchitobiose.
(11, 72), the L. acutangula lectin is notably better inhibited
The increase in the values of association constants (K,)
by tetra-N-acetylchitotetraose and penta-N-acetylchitopen- withligand size is apparentlynotrelatedto
a statistical
taose, implying an even more extended combining site than increase of binding probabilityfor the combining siteaccomin any lectin with related specificity. This lectin resembles modating asingle sugar residue, as the magnitude of the
marrow lectin and
WGA in its ability recognize
to
the internal affinitiesismuch
higher thanwhat could be ascribedto
di-N-acetylchitobiosyl sequences in the N-linked
glycopep- statistical effects (80). Alternatively, this consistently large
tides from fetuin, ovalbumin, and
soybean agglutinin (11, 35,
74, 75). Higher activities of the glycopeptides could be due to
We have also examined the binding of soybean agglutinin glycoadditional interactions with the sugar residues in the branchpeptide to the L.acutarzgulalectin by fluorescence spectroscopy where
ing regions or due t o a better orientation of the appropriate the extrapolated change in fluorescence intensity for totally bound
amounts to 16% with a 2 nmblue shift in the
di-N-acetylchitobiosyl conformer in these complex carbohy- glycopeptide [Om]
drates. However, the possibility of multiple interactions, also emission maximumin the fluorescence of the lectin. The values of K.
23,28, and 36 “C are 2.29 X IO5 M-’, 1.995 X lo5 M-’,and 1.349 X
termed as “cooperative effect” as observed for WGA (76, 77) at
IO5 M”, respectively. These values are quite close to the values
may be ruled out to explain the stronger binding
of L. acutan- obtained for penta-N-acetylchitopentaose.The values of -AH and
gula agglutinin to these glycopeptides, as the soybean agglu- -AS at 23 “C are 32.5 k J mol” and 7.1 J mol” K”, respectively. A
tinin glycopeptide, which is devoid of GlcNAc residues in the more favorable entropy when compared to the values obtained for
chito-oligosaccharides is presumably responsible for the strong bindbranching region, is an equally potent inhibitor when coming of the glycopeptide and may reflect a better juxtapositionof the
pared with theglycopeptides from fetuin andovalbumin. The glycopeptide
determinants for the binding to the lectin. No enhancerecognition of these glycopeptides by the lectin appears to bement was found on addition of soybean agglutinin AsN-oligosacchaexclusively mediated by the core di-N-acetylchitobiosyl seride (5 PM) and 14C-labeled Asn-GlcNAc(10 NM) at concentrations
indicated in parentheses. However, since no thermodynamic results
quence in these glycopeptides. This is also consistent with
our failure t o observe the inhibition of agglutination by the are available in the literature to compare the binding of glycopeptides
oligosaccharidederived
from endo-/3-N-acetylgIucosamini- to L. acutarzgula lectin, this phenomenon deserves greaterattention.
The fact that this lectin does not bind to N-1-[1-deoxy-N,N’dase H treatment of soybean agglutinin glycopeptide. T h e diacetylchitobiitol]-aminoethyl-BiogelP-300 (78), an affinity matrix
residues on either sideof the di-N-acetylchitobiosylsequence with a high density of P-D-G~CNAC
residues, also supports
the conclusion that multiple interactions involving bindingto monosaccharides
by themselves do not, therefore, contribute appreciably to the
alone are not sufficient to stabilize the complex.
V. Anantharam, and A. Surolia, unpublished observations.
Portions of this paper (including “ExperimentalProcedures,”
?Among the 3 tryptophanresidues in the protein onlyone is
“Results,” Figs. 1-6, and Tables I-VI) are presented in miniprint at
the end of this paper. Miniprint is easily read with the aid of a accessible to N-bromosuccinimide when the reaction is carried out
standard magnifying glass. Full size photocopies are available from under native conditions.The modification of this tryptophan residue
the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, located on the surface of the lectin completely abrogates its carboMD 20814. Request Document No. 85M-2245, cite the authors, and hydrate-binding ability(V. Anantharam andA. SuroIia, unpublished
include a check or money order for $7.20per set of photocopies. Full observations).The ligand-induced alterations in the emission spectra
size photocopies are also included in the microfilm edition of the of the lectin can therefore be ascribed to the Perturbation of the
surface tryptophan residue which is also involved in its activity.
Journal that is available from Waverly Press.
EXPERIMENTALPROCEDURES AND RESULTS3
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A Tetra-N-acetylchitotetraose-specific
Exudate Lectin
TABLE
VI1
Proposed arrangement of chito-oligosaccharides in various subsites of
L. acutangula lectin and AG and AH contributions of each subsite
for the ligand binding deduced from Table VI
These @ ( 1 4 ) - l i n k e dsaccharides are arranged in their respective
subsites with the reducing end to theright. The contribution of each
subsite to the association of an oligosaccharide with L. acutangula
lectin was calculated by comparing AG and AH values for pairs of
saccharides which differ by a single sugar residue. It isassumed that
an additional sugar residue (italicized in the table) is bound a t the
subsite listed in the column with its contribution to AG and AH as
determined by comparing the values for pairs of saccharides.
Contribution to
A
B
C
D
E
-AG
17.0
5.3
G ~ ~ N A ~ - G ~ ~ N A ~ - G ~ c N A c - G ~ N 5.4
Ac
5.1
GlcNAc-GlcNAc-GlcNAc-GlcNAc-GlcNAc
enthalpy is insignificant. However, a comparatively low value
of - A S for penta-N-acetylchitopentaose suggests that the
binding of this saccharide is highly favorable. The values of
-AS, which are larger than theentropies of mixing (511, also
show a progressive increment asthe number of GlcNAc units
in thesaccharides increase (up to the
tetrasaccharide). These
unfavorable changes in entropy conceivably reflect loss in
translational entropy and/or changes in conformationts) of
the saccharides and/or the lectin. Nevertheless, the binding
of saccharides to L. acutangula lectin is highly favorable
because of an increase in thevalues of -LW which outweighs
the unfavorable contribution of change in entropy.
-AH
kJ mol"
GlcNAc
GlcNAc-GkNAc
GlcNAc-GlcNAc-GkNAc
14623
41.0
6.9
8.0
0.1
Schematization of the binding positions represented here may be
an oversimplification of the binding process; however, the increase in
the fluorescence intensity and theblue shift accompanying the ligand
binding have allowed us to prefer the saccharide locations on various
subsites as outlined in TableVII.
Addendum-The values of the association constants for the binding of hexa-N-acetylchitohexaose(from Seikagaku Kogyo Co., Ltd.,
Tokyo, Japan) to L. acutangula lectin are 1.13 X lo6 "I, 7.5 X lo6
"I,
5.3 X lo6 "I, and 2.75 X lo6at 15,20,25,and 36 "C,respectively.
The values of -AG, - A H , and -AS correspond to 32.67 kJ mol", 55
kJ mol", and 75 J mol" K" at 25 "C, respectively.
A lack of increment in the value of -AH for the binding of hexaN-acetylchitohexaose when compared with tetra-N-acetylchitotetraose is consistent with our proposal that thecombining region of
L. acutangula lectin is most complementary toward tetra-N-acetylchitotetraose.
REFERENCES
1. Lis, H., and Sharon,N. (1973) Annu. Reu. Biochem. 42,541-574
2. Liener, I. E, (1976) Annu. Rev. Plant Physiol. 27,291-319
3. Goldstein, I. J., and Hayes, C. E. (1978) Adu. Carbohydr. Chem.
Biochem. 35,127-340
4. Pereira, M.E. A., and Kabat, E. A. (1979) Crit. Rev. Immunol. 1 ,
33-78
5. Barondes, S. H. (1981) Annu. Reu. Biochem. 50, 207-231
6. Surolia, A., Bachhawat, B. K., and Podder, S. K. (1975) Nature
257,802-804
7. Surolia, A., Bishayee, S., Ahmed, A., Balasubramanian, K. A.,
Thambidorai, D., Podder, S. K., and Bachhawat, B. K.(1975)
in Concanavalin A (Chauduri, T. K., and Weiss, A. K. e&) pp
95-115, Plenum Press, New York
8. Podder, S. K., Surolia, A., and Bachhawat, B. K. (1974) Eur. J.
Biochem. 44,151-160
9. Sequiera, L. (1978) Annu. Rev. Phytopathl. 16,453-481
10. Sabnis, D. D., and Hart,J. W. (1978) Planta 142,97-101
11. Allen, A. K. (1979) Bioehem. J. 183,133-137
12. Greenaway, P. J., and LeVine, D. (1973) Nat. New Biol. 241,
191-192
13. Allen, A. K., and Neuberger, A. (1973) Biochem. J . 135,307-314
14. Kilpatrick, D. C., and Yeoman, M.M. (1978) Biochem. J. 1 7 6 ,
1151-1153
15. Nachbar, M. S., Oppenheim, J. D., and Thomas, J. 0.(1980) J.
Bwl. Chern. 255,2056-2061
16. Kilpatrick, D. C. (1980) Biochem. J. 1 8 5 , 269-272
17. Crowley, J. F., Goldstein, I. J., Arnarp, J., and Lonngren, J.
(1984) Arch. Biochem. Biophys. 231, 524-535
18. Monsigny, M. A., Roche, A.C., Sene, C., Moget-Dana, R., and
Delmotte, F. (1981) Eur. J. Biochem. 104,147-151
19. Shibata, S., Goldstein, I. J., and Baker, D. A. (1982) J. Biol.
Chem. 257,9324-9330
20. Majumdar, T., and Surolia, A. (1978) Prep. Bwchem. 8, 119-131
21. March, S. C., Parikh, I., and Cuatrecasas, P. (1974) Anal.
Biochem. 60,149-152
22. Roseman, S., and Ludowieg, J. (1954) J . Am. C h e n . Sac. 7 6 ,
301-302
23. Rivers, R. P., and Neuberger, A. (1939) J . Chem. Sac. (Lond.) 1 ,
122-126
24. Allen, A. K., Davies, R. C., Neuberger, A., and Morgan, D. M. L.
Downloaded from www.jbc.org by on June 16, 2008
increase in the K,, values for the binding of the lectin to
saccharides with increase in their size can be explained if one
assumes that the combining site of the lectin consists of a
number of subsites, each of which accommodates one sugar
unit, and that theassociation of a given subsite with a single
sugar moiety is independent of other units in the saccharide.
With the assumption that the-AG for any saccharide equals
the sum of the free energies from each subsite filled, the
contribution of -AG from each subsite inthe lectin is obtained
by comparing a pairof saccharides, and these values, together
with the probable positions for the binding of saccharides, are
listed in Table VII. B, C, and D are strong binding subsites,
E is a moderate affinity subsite, and subsite A is unfavorable,
since GlcNAc does not bind. The fact that there is a striking
increase in -AG of binding as additionalunits of GlcNAc are
added would support our view that B-C-D-E are contiguous
favorable subsites.'
The thermodynamic parameters obtainedfrom the temperature dependence of K , values differ appreciably for oligosaccharides of different length (Table VI). Subsites C, D, and E
contribute 6.9, 8.0, and 0.1 kJ mol", respectively, to the
binding enthalpy of the corresponding GlcNAc residues of the
saccharides. In light of these data, we consider this lectin to
possess an extended binding site which accommodates a tetrasaccharide. This is in marked contrast to concanavalin A,
where the values of -AH remain constant with the increase
in the chain lengthof cu(l-+2)-linked manno-oligosaccharides
(81).In WGA, the values of -AH are ligand chain lengthdependent upto thetrisaccharide; however, the effect is larger
for the disaccharide di-N-acetylchitobiose over that observed
for GlcNAc (82). It is interesting to compare here the situation
of L. acutangula lectin with lysozyme, where the bindings of
GlcNAc, di-N-acetylchitobiose, and tri-N-acetylchitotriose
show a pronounced increase in -AH with increase in ligand
chain length (83, 84). Likewise, astriking increase in the
values of -AH would imply more extensive hydrogen bonding,
and van der Waal's interactions in the binding site of L.
acutangula lectin as the dimensions of the saccharide(s) are
increased. Practically unchanged values of -AH for penta-hracetylchitopentaose when compared with tetra-N-acetylchitotetraose indicate that contribution of subsite E to binding
Acknowledgments-We thank G. V. Swarnalatha for her technical
assistance, Prof. P. Balaram for the use of Spectrofluorimeter, Dr. S.
K. Brahmachari for the use of analytical ultracentrifuge, and Dr. M.
Bansal for the EMBL-DA 05 computer program written by S. W.
Provencher used for the determination of secondary structure from
CD. Sodium b~ro[~H]hydride
was a generous gift from Prof. Subhash
Basu, University of Notre Dame. We thank Mary Maliarik for independently checking the purity and molecular weight of the protein.
14624
A Tetra-N-acetylchitotetraose-specific
Exudate Lectin
Supplement t o I J o l a t i o n
Hacrmolecular P r o p e r t i e sa n dC o m b i n i n gS i t e
of a
C h i t P - e l i g o s a C C h a r i d eS b e c i f i cL e c t i nf r o mt h eE x u d a t eo fR i d g e
Gourd
(Luffa a c u t a n q u l e ) .
VellareddyAnantharam,Sankhavdram
R. P a t a n j a l i , M. Joginadha Swamy,
Ashok R. S a n a d i , I r w i n J. GoldsteinandAvadherha
Surolia.
ExDerimentalProcedures
Materials:Sephazose-68,
SephadeX GlW, G 5 0 . G 2 5 , G 1 5 and GI0 were
p r o d u c t eo fP h a m a c i a ,U p p a a l a ,
Sweden. P r o t e i n su s e d
as molecular w e i g h t
as well as SDS molecular weightmarkers,
mannosaminehydrochloride
N-Lset l l a c t o s a m i n ec h i t i n f. e t u i n .
concanavalin
-ex
A (con A) *eat
g e m a g g i u t i n i n Iwa] o v a l b u hnne u r a m i n i d a s e .
of sigma c h e m i c a l
oarex 1-x8 and ~ a n s y lamino'acidswere'products
5c+x8,-x2:
Co., S t .L o u i s .
U.S.A.
Endo-8-N-acetylglucoraminidase H was a p r o d u c to f
wae p r e p a r e d as d e s c r i b e d In
K o c h - L i g h tL a b o r a t o r i e s .S o y b e a na g g l u t i n i n
(20).
Con A and WGA wereCoupled t o Sephamse-68bythemethod
of March
e t a l . ( 2 1 ) . Sodium b o r o [%] hydride. ['251] NaI and[14C]
forfnaldehyde
were p r o d u c t s of h e r s h a mC o r p o r a t i o n .~ l l i n o i s
U S A
R a d i o a c t i v e measurements Wedone using 1275 'Mini G-a
gamma &t;r'Model
43. LKB (WallaC)
andon LKB [ l l a l l a c ]1 2 1 7 Rack b e t s l i q u i d - s c i n t i l l a t i o n c o u n t e r , w i t h t h e
aidofscintillationfluid
made up by d i s s o l v i n g 800 mg ofdlphenyloXazO1e
and 20 mg of 1.4-bis[5-ph;nylorar~le-2-y1]benzenein
ZM) m l o ft o l u e n ea n d
100 m l o fT r i t o n X-100.
All o t h e rC h e m i c a l s were comercia1 p r o d u c t s Of t h e
were bought
h i g h e s tq u a l i t ya v a i l a b l e .R i d g eg o u r d[ L . a c u t a n ( l u l a ]f r u i t s
from local green grocers.
markers in gel chromatography
MeChodq
P r e p a r a t i o n ofN-acetylmannoramine.
M e t h y l - Z - a s e t ~ i d o - Z - d e o x y a and P-CglucoDVrenOaides: Mannosamins h y d r o c h l o r i d e Was N-acetylated as d e s c r i b e d
I n (22).
Anomerr o fm e t h y l GlcNAc were p r e p a r e d as d e s c r i b e d i n (23.24).
oliamsrs andmann~oli.oracchar1d.s:ChitinhydroP r e n a r a t i o no fc h i t i n
The c h i t i n oligomers Were
l y s a t e was p r e p a r e d as d e s c r i b e d by Rupley (25).
s e p a r a t e d on Biogel P-2 ( 2 6 ) and t h e i r p u r i t y Was a s s e s s e d by t h i n layer
s[1->2]
l i n k e d M a n n o - ~ l i g o ~ a c c h a r i d e were
r
chromatography (27).
prepared
a$ d e s c r i b e d i n ( 2 8 ) .
55. Mann, K. G., Nesheim, M. E., and Tracy, P. B. (1981) Bwchemistry 20,28-33
56. Tanford, C. (1961) Physical Chemistry of Macromolecules, p. 359,
Wiley-Interscience, New York
57. Gray, W. R. (1972) Methods Enzymol. 25, 121-138
58. Woods, K.R., and Wang, K. T. (1967) Biochim. Biophys. Acta
133,369-370
59. Spencer, R. L.,and Wold, F. (1969) Anal. Bwchem. 32, 185-190
60. Hoy, T. G., Ferdinand, T. G., and Harrison, P. M. (1974) Znt. J.
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61. Barman, T. E., and Koshland, D. E., Jr. (1967) J. Bbl. Chem.
242,5771-5776
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63. Schachman, H. K. (1957) Methds Enzymol. 4,32-104
Biochemistry
64. Marler. E.. Nelson. C. A., and Tanford.. C. (1964)
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3,279-284
65. Lis. H.. and Sharon. N. (1972) Methods Enzvmol. 28.360-365
66. Scatchard, G. (1949)Ann. N . 'Y. Acad. Sci. 51, 660-672
67. Provencher, S. W., and Glockner, J. (1981) Biochemistry 20,3337
68. Provencher, S. W. (1982) Technical Reportof European Moleculnr
Biology Laboratory-DA 05,Heidelberg, West Germany
69. Chipman, D. M., Grisaro, V., and Sharon, N. (1967) J. Biol.
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70. Jirgensons, B. (1978) Biochim. Biophys. Acta 536,205-211
71. Longren, J., Goldstein, I. J., and Zand, R. (1976) Biochemistry
15,436-440
72. Matsumoto, I., Jimbo, A., Mizuno, Y., Seno, N., and Jeanloz, R.
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73. Allen, A. K., Neuberger, A,, and Sharon, N. (1973) Biochem. J.
131, 155-162
74. Goldstein, I. J., Hammerstrom, S., and Sundblad, G. (1975)
Bwchim. Biophys. Acta 405, 53-61
75. Spiro, R. G. (1973) Adu. Protein Chem. 27,349-457
76. Bhavanandan, V. P., Umemoto, J., Banks, J. R., and Davidson,
E. A. (1977) Biochemistry 16,4426-4437
77. Bhavanandan, V. P., and Katlic, A.W. (1979) J. Biol.Chem.
254,4000-4008
78. Baues, R. J., and Gray, G. R. (1977) J. Bwl. Chem. 252,57-60
79. Lotan, R., and Sharon, N. (1973) Bwchem. Bwphys. Res. Comnun. 55, 1340-1346
80. Brewer, C. F., and Brown, R. D. (1979) Biochemistry 18, 25552562
81. Van Landschoot, A., Loontiens, F. G., and De Bruyne, C. K.
(1980) Eur. J. Biochem. 103, 307-312
82. Van Landschoot, A., Loontiens, F. G., Clegg, R. M., Sharon, N.,
and De Bruyne, C. K. (1977) Eur. J. Bwchem. 79,275-283
83. Bjurulf, C., Laynez, J., and Wadso, I. (1970) Eur. J. Biochem.
14.47-52
84. Banerjee, S. K., and Rupley, J. A. (1973) J. Bwl. Chem. 248,
2117-2124
A n a l y t i c a lM e t h o d s
r-"----~
-
~
~
..
.
a c i d analyzer Model LKB 4400 a f t e r h y d r o l y s i s With 4N HCl a t 1OooC f o r 6h.
N e u t r a l sugar was also determined by h y d r o l y z i n g t h e g l y c o p e ~ t i d e i n 1 N HCl
for 4h a t lW0C i ns e a l e d andevacuatedtubes.Afterthe
removal Of a c i d .
t h e h y d r o l y s a t e was passedthroughCoupled
columns Of Dowel 5C-X8 (H+ form)
andDwex 1-X8 ( a c e t a t ef o r m )
(30). The n e u t r a l lugsrs t h u se l u t e d were
d e t e c t e d and q u a n t i f i e d by p h e n o l - s u l f u r i ca c i dm e t h o d .A s p a r t i ca c i d
from
g l y c o p e p t i d e s i n coIumII Chromatographyexperiments was d e t e c t e db yn i n h y d r i n
and
r e a c t i o n (31)
Paper chromatography f o r n e u t r a l sugars hexosaminel
oligosacchari;(es %ere c a r r i e d Out on matman N0.1 p a p a r ' b yt h ed e s c e n d i n g
was p y r i d i n e - e t h y l a c e t a t ~ - = t ~ ~ = c ~ t i c
t e c h n i q u ea n dt h es o l v e n tS y s t e mu s e d
a c i d (5:5:3:1)
w i t h pyridine-ethyl.cetdte-uater
(11:40:6) i n t h eb o t t o mo f
thechromatographychamber
(32).
P r o t e i nc o n c e n t r a t i o n s
were d e t e r m m e db y
(33) or by t h e i r a b s o r b a n c e a t 280 m.
thedye-bindingmethodofBradford
h i n o sugars were q u a n t i f i e d as p e r t h e p r o c e d u r e
given i n (34).
~~
as p e rt h ep r o c e d u r eo fL i s
and Sharon ( 3 5 ) w i t h minor m o d i f i c a t i o n s as
d e s c r i b e d b e l w in b r i e f . Soybean a g g l u t i n i n (500 mg) 118s d i s s o l v e d in 80 m l
of 0.1 M HC1 and i t s pH a d j u s t e d t o 2. The p r o t e i n Was d e n a t u r e db yh e a t i n g
t o pH 8.5 w i t h 1 M
thesolutionfor
15 m i " a t 50°C t h e pH was t h e n b r o u g h t
NaW. 10 t h i s s o l u t i o n , proms: (Calbiochem) (5 mg) i n 5 m l of Iris b u f f e r .
for 72 h a t
pH 8.5 and 0.06 M in CaCl,. was a d d e da n dt h em i x t u r ei n t u b a t e d
Downloaded from www.jbc.org by on June 16, 2008
(1976) Carbohydr. Res. 51,149-155
25. Rupley, J. A. (1964) Biochim. Biophys. Acta 83,245-255
26. Raftery, M. A., Rand-Meir, T., Dahlquist, F. W., Parsons, S. M.,
Borders, C. L., Jr., Wolcott, R. G., Beranek, W., Jr., and Jao,
L. (1969) Anal. Biochem. 30,427-435
2 7. Powning, R. F., and Irzykiewicz, H. (1967) J. Chromutogr. 29,
115-119
28. Kocourek, J., and Ballou, C. E. (1969) J. Bacteriol. 100, 11751181
29. Dubois, M., Gillis, K.A., Hamilton, J. K., Rebers, P. A., and
Smith, F. (1956) Anal. Biochem. 28, 350-356
30. Spiro, R. G. (1966) Methods Enzymol. 8, 3-26
31. Moore, S., and Stein, W. H. (1954) J. Bwl. Chem. 211,907-913
32. Fischer, F. G., and Nebel, H. J. (1955) Hoppe-Seyler's2.Physiol.
Chem. 302, 10-19
33. Bradford, M. M. (1976) Anal. Biochem. 72,248-254
34. Good, T. A., and Bessman, S. P. (1964) Anal. Biochem. 9, 253262
35. Lis, H., and Sharon, N. (1978) J. Bwl. Chem. 253,3468-3476
36. Dorland, L., van Halbeck, H., Vliegenthart, J. F. G., Lis, H., and
Sharon, N. (1981) J. Biol. Chem. 256, 7708-7711
37. Warren, L. (1963) Methods Enzymol. 6, 463-465
38. Li, Y. T., and Li, S. C. (1972) Methods Enzymol. 28,702-713
39. Spiro, R.G., and Bhoyroo, V. D. (1974) J. Biol.Chem. 2 4 9 ,
5704-5717
40. Nilsson, B., NordLn, N. E.,and Svensson, S. (1979) J. Bwl. Chem.
254,4545-4563
41. Tai, T., Yamashita, K., Ogato-Arakawa, M., Koide, N., Muramatsu, T., Iwashita, S., Inoue, Y., and Kobata, A. (1975) J.
Biol. Chem. 250,8569-8575
42. Tai, T., Yamashita, K., Ito, S., and Kobata, A. (1977) J. Biol.
Chem. 252,6687-6694
43. Yamashita, K., Tachibana, Y., and Kobata, A. (1978) J. Biol.
Chem. 253,3862-3869
44. Rice, R. H., and Means, G. E. (1971) J. Biol. Chem. 246, 831832
45. Tarentino, A. L., and Maley, F. (1974) J. Biol. Chem. 2 4 9 , 818824
46. Tarentino, A. L., Trimble, R. B., and Maley, F. (1978) Methods
Enzymol. 50, 574-580
47. Weber, C., Franke, W. W., and Kartenbeck, J. (1974) Elcp. Cell
Res. 87, 79-106
48. Riesfeld, R. A., Lewis, U. J., and Williams, D. E. (1962) Nature
195,281-283
49. Laemmli, U. K. (1970) Nature 227,680-685
50. Weber, K., and Osborn, M. (1969) J. Biol. Chem.244,4406-4412
51. van Holde, K. E. (1971) PhysicalBiochemistry, pp. 16, 105,
Prentice-Hall, Englewood Cliffs, NJ
52. Yphantis, D. A. (1964) Biochemistry 3,297-317
53. Andrews, P. (1970) Methods Biochem. Anal. 18,26-36
54. Ackers, G. K. (1964) Biochemistry 3,723-730
A Tetra-N-acetylchitotetraose-specificExudate Lectin
P r e p a r a t i o n of a s i a l o f e t u i n and a a a l a s t o f e t u i n :F e t u i n
was d e s i a l a t e d by
h e a t i n g a t BO'C i n 0.011 N &SO,.
The e x t e n t o f d e s i a l a t i o n
was determined by
t h i o b a r b i t u r i ca c i dm e t h c d
( 3 7 ) . A s i a l o f e t u i n ( 5 0 3 mg i n3 0 m l ) was incui n 0.15 M s o d i m c i t r a t e b u f f e r a t
b a t e dw i t h8 - a a l a c t o s i d a a ef r o mJ a c k b e a n
120 h a t 37% ( 3 8 ) . The r e l e a s e d
pH 4.5 a t 1 u n i t / m lc o n c e n t r a t i o nf o r
g a l a c t o s e was q u a n t i f i e d by n e u t r a l sugar c o n t e n t a f t e r i t s r e p a r a t i o n
from
5 0 and Dowel 1 c o l m n and c h a r c a s l r e a c t i o n m i x t u r e using t h ec o u p l e d 0-x
ce1ite C O l r n .
P r e p a r a t i o n of b l i n k e dm l v c o v e p t i d e sf r o mf e t u i n .a s i a l o f e t u i na n d
aqlllaEtOfetUin:N-linkedglycopeptides
were prepared by t h ep r o c e d u r e
Of
s p i r o andBhoyroo
( 3 9 ) . F e t u i n ( 2 g ) , a s i a l a f e t u i n ( 2 9 ) a n da g a l a c t o f e t u i n
( 4 0 0 mg) were i n c u b a t e d s e p a r a t e l y w i t h
0.8 M NaBH4 i n 0.1 M NaOH a t 3TaC
f o r I 2 h. The r e a s t i o n m i x t u r e was b r o u g h t t o pH 5 . 0 by slwr e d i t i o n of 4N
a c e t i ca c i d .
The sample was t h e nt h o r o u g h l yd i a l y s e d .F e t u i n .a s i a l o f e t u i n
a n d a g a l a c t o f e t u i n were t h e n s u b j e c t e d t o p r o n a s e d i g e s t i o n e t
37% in
0.15 M T r i s a c e t a t e b u f f e r
pH 7 8 c o n t a i n i n g 1.5 mM c a l c i u ma c e t a t e .
again 0.5% (w/w) was added
I n i t i a l l y t h e 1* ( w h ) of enz& ;as addedand
a f t e r 24 hand 48 hr e s p e c t i v e l y .A f t e r9 6h ,l y o p h i l i z e dp r o n a s ed i g e s t
was di*sOlved i n 0.1 M p y r i d i n e a c e t a t e b u f f e r a t
pH 5.0 and was f r a c t i o n a t e d i n t h i s b u f f e r on Sephadex Cr50 t o remove p a r t i a l l y d i g e s t e d p r o t e i n .
The g l y c o p e p t i d e se m e r g i n g
i n t h e i n n e r vollans of Sephadex G50 column were
d e t e c t e db yn e u t r a l
SYgaP a n a l y s i s .
The g l y c o p s p t l d ec o n t a i n i n gf r s c t i o m
w e n p o o l e da n da g a i ns u b j e c t e d
to pronase d i g e s t i o n a n d P e c h r m a t o g r a p h e d
On Sephadex Cr50 as mentioned above.
T h eg l y c o p e p t i d e t h u s o b t a i n e d Va6
w r i f i e d On Senhadex G25 and t h e o e a k e l u t i n a i n t h e v o i d volume Was chai a c t e r i r e d f o r ' n e u t r a l sugar, aminb suga; a n d - m i n oa c i d s as s t a t e d i n t h e
h a 1 t i c a l m e t h o d s .T h es t r u c t u r e
of t h ef e t u i ng l y c o p e p t i d e
(40) is g i v e n
i n Frgure 1.
Ovalbumin (100 mg/4 m l ) was s u b j e c t e d t o two c y c l e s o f p r o n a s e d i g e s t i o n i n 0.15 u T r i r a c e t a t e b u f f e r p ~
1.8 a t 37%.
I n i t i a l l y I$ (w/w)
again O.%
(w/w) waa added a t 24 hand48h
resOf enzyme We* addedand
PoCtiVely.After
96 h .t h el y o p h i l i z e dp r o n a s ed i g e s t
was d i s s o l v e di n
0.1 M p y r i d i n e a c e t a t e b u f f e r a t
pH 5.0 and f r a c t i o n a t e d i n t h e same b u f f e r
on Sophsdex G50. T h eg l y c o p e p t i d e se l u t e d
in t h e i n n e r volume ofSephadex
6-50 colwere c h a r a c t e r i z e d for t h e i r C h e m i c a l E m p o s i t i o n as above and
t h e m i X t U r 0 Of g l y c o p e p t i d e 6 was u s e df o rf u r t h e rs t u d i e s .
The S t N C t U m
of theovalbuminglyCOPePtide8
(CW-GP) am given i n (41-43).
14625
S e d i m e n t a t i o nV e l o c i t va n de q u i l i b r i u mm e a s u r e m e n t i :S e d i m e n t a t i o nv e l o c i t y
mea8urements were performed a t 41000 r p m w i t h Beckman LB-70 u l t r a c e n t r l f u g s
neze c a r r i e dO u t a t
e q u i p p o dw i t h a p h o t o e l o c t r i e ScaDnez.Theexperiments
ZO°C i n p r e s e n c e a n d a b s e n c e o f
1% pME and 6 M g u a n i d i n e h y d r o c h l o r i d e
(GuHC1).
P r o t e i nC o n c e n t r a t i o n s In t h e range of 0.6 t o 1.0 mghl were used.
The s e d i m e n t a t i o n c o e f f i c i e n t s (&.)
were c a l c u l a t e d as d e s c r i b e d i n
van Hold. (51). S e d i m e n t a t i o ne q u i l i b r i u ma n a l y s i s
was p e r f o m e dw i t h
SPinCO Model E analytical u l t r a c e n t r i f u g e e q u i p p e d W i t h
a photoClestriC
scanner. La, Speed (11.270 m) e q u i l i b r i u ms e d i m e n t a t i o n was c a r r i e d out
a t 20°C.
For the,. s t u d i e s n a t i v e l e c t i n
($so
1.0) was d i 8 s o l v e d i n PBS
With and without
ly @ME d i a l y z e d e x t e n s i v e l y a g a i n s t t h e
same b u f f e r t o b e
used as reference. The m o l e c u l a rW e i g h t Was d e t e r m i n e d as described by
Yphantis(52).
-
M1 f i l t r a t i o n :
Gel f i l t r a t i o n was p e r f o r n e d on Sophadex GloD i nt h e
end g r o u pa n a l y s i s :Q u a l i t a t i v eN H 2 - t e m i n a le n d
NH2-tenninal
group a n a l y s i s
was c a r r i e do u tb yt h ed a n s y l a t i o nm e t h o dm o d i f i e df o rp r o t e i n s
as d e s c r i b e d
by Gray ( 5 7 ) . W 2 - t e r m i n a l amino a c i d was i d e n t i f i e d by chromatography on
Polyamide layers (58)usingDansylaminoacid5
as s t a n d a r d .
4 i n o a c i d COmDOSitiQn: P r o t e i ns a m p l e s for amino a c i d a n a l y s z s were conc e n t r a t e d i n vacuo. h v d r o l y s e d i n 6 . 0
N HC1 a t llO°C f o r 2 4 h u n d e r n i t r w e n
and a n a l y s e d on a &&an
i2cc aminoacid analyser. Amino a c i d a&yses
were c a r r i e do u tu s i n gt h es i n g l e - c o l u m n .t h r e eb u f f e rs y s t e m
(Durham
C h e m i c a lC o r p o r a t i o n ) .
The l e c t i n was also hydrolysed i n t h ep r e s e n c e of
d i m e t h y l s u l f o x i d et od e t e r m i n ec y s t e i n er e s i d u e s( 5 9 ) .
The amino a c i d c o r n
p o s i t i o n was d e t e r m i n e d u s i n g a computerprogramof
Hoy e t al. (60)
phsnContent was determinedaccording
t o t h e method of BarmanandK&hl%te
8.0 M urea. T o t a lt r y p t o p h a n was
( 6 1 )b o t hi nt h ep r e s e n c e
and absenceof
a l s o d e t e r m i n e da c c o r d i n gt ot h em e t h o do fE d e l h o c h( 6 2 ) .
The p a r t i a ls p e c i Schachman ( 6 3 ) . G i nb u f f e r
f i c volume (?) was c a l c u l a t e da c c o r d i n gt o
c o n t a i n i n g 6 M GuHCl was assumed t o b e 0.69 m l g-', as a d i f f e r e n c e of 0 . ~ 2
i s e x p e c t e di nt h a ts o l v e n t
(64).
~
SBA-OS
F.1-GP
581c-05. S E A
oligosaccharide;
Fete,fetuin
glycopeptide;
ASF-GP. a s i a l o fetuin.glyccp e p t i d e ; IYiF-GP.
aqalactafetuio
glycopeptide;
Ovalbunin
g l y c o p e p t i d e . In
all c a s e st h e core
s t r u c t u r e is t h e
following:
lal,
6[1=1,31 Mkl,
4 W ? , 4GBN. All
peripheral g a l
and GlCNAc l i n kages are 8 . r e s t
G-N
o ft h el i n k a g e s
are B . Arabic
numerals i n d i c a t e
t h e linkage p o s i t i o n t o t h e u n d e r l y i n g
sugar when shown. For d e t e r m i n i n g
sugar were t a k e n a$ r e f e r e n c e f o r glycot h e r a t i o s . amino acidandamino
p e p t i d e sa n d
Oligosaccharides r e s p e c t i v e l y .
*In case o fo v a l b m i nt h e
m i x t u r eo fg l y c o p e p t i d e s
were u s e d W i t h o u t f u r t h e r p u p i f i c a t i o n .
ASF-GP
OV-GP.
P r e p a r a t i o n of S a y b e a nA g g l u t i n i na n dA s i a l o f e t u i n
[14c1 mn qlYCoPeDtides
and ASF-GPb
SBA-72 [l mg a$ mannose] and ASF-GP 11.5 mg a$ n e u t r a l
sugar] were l a b e l l e dW i t h [ 1 4 C ] formaldehyde ( 3 pmol, 30 mCl/moll indepenRlce and Means ( 4 4 ) . The l a b e l l e dg l y c o p e p t i d e s
d e n t l y by themethodof
were p u r i f i e d by gel f i l t r a t i o n on Sephadex G l O .
LSBA-GP
T r e a t m e n to fr 1 4 C ll a b e l l e d
SBA-GP and ASF-GF w i t h e n d c - 8 - N - a c e t . l u c e s a m i n i d a s e H: [14C] l a b e l l e d SEA-GF [ 3 W ilgs as m a m o J e . c o n t a i n i n g
2.2 x lo5 epml was mixedwith an e q u a l amountof
u n l a b e l l e d SBA-GF i n I m l
Of 0.1 M a c e t a t e b u f f e r
pH 5 . 5 .
T h i s was t h e nI n c u b a t e dw i t h
0.2 u n i t s of
e n d o - P - N - a c e t y l g l u c o ~ ~ m i n i d a s cH f o r 20 h a t 37'C ( 4 5 ) . The r e a c t i o n was
s t o p p e d by h e a t i n g f o r 1 min i n a b o l l i n g w a t e rb a t h and t h e r e a c t i o n m i x t u r e
w a s f r a c t i o n a t e d en Sephadex 6 1 5 i n 0.01 M a c e t i c a c i d
where 2 peaks con11 which
t a i n i n gr a d i o a c t i v i t y were o b s e r v e d .H i g h e rm o l e c u l a r& e i g h t[ p e a k
was pooled and chromatographed
had 1%. r a d i o a c t i v i t y o f t h e s t a r t i n g m a t e r i a l
on D w e x 5 0 x 2 e q u i l i b r a t e d w i t h 1 mhl p y r i d i n e a c e t a t e b u f f e r
pH 2 . 7 , and
sugar Which was d e v o i do fr a d i a a c t i v l t y
was
t h ep e a kc o n t a i n i n gn e u t r a l
pooled.
Lack of r a d i o a c t i v i t yi nt h en e u t r a l
Pugar c o n t a i n i n gf r a c t i o n s
and GlcNAC-AsN havebeen
i n d i c a t e d t h a t a l l of t h eu n d i g e s t e dg l y c o p e p t i d e
s e p a r a t e df r o mt h eo l i g o s a c c h a r i d ef r a c t i o n .
[I4C] i a b e l l e d ASF-GP [ 5 0 0 kg as n e u t r a l suqar. 1.9 x lo5 cpm] wa5
also t r e a t e d w i t h 0 . 1 5 u n i t of e n d ~ - D - N - a c e t y l g l u c o s a m i n i d a s e H a n dc h i "
matographed on a Sephader 6 1 5 column where a s i n g l e p e a k c o n t a i n i n s r a d i b
s c t i v i t y was o b t a i n e d . The only f r a c t i o n o b t a i n e d was e l u t e d again- on
D W e x 5C-X2 w h e r et h ef r a c t i o n s
con t a i n m g r a d i o a c t i v i t y and n e u t r a l sugar
were c o i n c i d e n t .B a s e d
on t h e s e we presumethe I t ASF-GP h a sn o tb e e nc l e a v e d
by end a -8 -N -a c e ty l g l u c o r a m inidase H ( 4 6 ) .
P r e D a r a i i o n Of a f f i n i t y S U D D O r t :
Soybean a g c l u t i n i n g l y c o p e p t i d e (200 m g )
war Coupled t o cyanogen bromide activated Sepharaae-68(125
ml) i n 0.2 M
s o d i u mb i c a r b o n a t eb u f f e r
pH 9.0 f o r1 6 h a t 4OC ( 2 1 )A f t e rb l o c k i n gt h e
u n r e a c t e dg r o u p sf r o mt h e
gel and e x h a u s t i v eW a s h l n g . ' t h e
amount Of glycop e p t i d eC o u p l e d was e s t i m a t e d t o be 350 po/m1 gel.
P u r i f i c a t i o n of r i d g e 4ouPd l e c t i n : The b u f f e ru s e di n
a l l experiment3 was
0.02 M sodiumphosphate.
pH 7 . 4 .c o n t a i n i n g0 . 1 5
M NaCl and 10
d
l P-mercaptoe t h a n o l (PBS-PME).
All t h ep r o c e d u r e s
were c a r r l e do u ta t
4OC. F r u i t s were
b l e d( 4 7 )
and e x u d a t e was c o l l e c t e di n t o oxygen free b u f f e r . The b u f f e r
c o n t a i n i n g t h e e x u d a t e was t h e n c e n t r i f u g e d a t 9000 a g f o r 10 mi" a n d t h e
was p r e c i p i t a t e d by 65/ m o n i u m w l f a t e .
The
p r o t e i ni nt h es u p e r n a t a n t
PSS-$ME.
p r e c i p i t a t e was t h e n d i a l y s e d a g a l n s t
A f f i n i t yC h r m a t o a r a D h y :
The clear s u p e r n a t a n tf r o mt h ep r e v i o u ss t e p
was
loaded on s o y b e a n a g g l u t i n i n glycopeptide-Se~harose-68 column e q u i l i b r a t e d
w i t h PBS-@UE. The Column 1 1 8 w a s h e dw i t ht h e
6 m e b u f f e r t i l l no more
p r o t e i nc o u l db ed e t e c t e d
in t h e washings (h2a0 < 0.005). The bound p r o t e i n
Hemaaqlutination a s s a y : The a g g l u t i n a t i o na c t i v i t y was a s s a y e da c c o r d i n gt o
t h ep h o t o m e t r i cm e t h o d
of L i s andSharon(65).
The l e c t i n was s e r l a l l y
d i l u t e d i n 0.5 ml o f s a l i n e i n
10 x 7 5 m t e s t t u b e s .
To e a c ht u b e was added
0.5 m l o f s t a n d a r d r a b b i t e r y t h r o c y t e s u s p e n s i o n
s o t h a t an absorbance Of 1
a t 6 2 0 nm was o b t a i n e d .A f t e r
2 ht h ea b s o r b a n c e
a t 620 m was recorded
One h e m a g g l u t i n a t i o nu n i t [ W ] is d e f i n e d &I t h ea m o u n to ft h el e c t i n
[l'pg]
which is r e q u i r e d t o cause a 50/ d e c r e a s e i n t h e a b s o r b a n c e of e r y t h r o c y t e
SusPension i n 2 h.
For 1 W A a g g l u t i n a t i o n [%20 < 0.071theamountofthe
l e c t i nr e q u i r e di nt h e
a s s a y i n 2 kg/rnl.
F o ri n h i b i t L o ns t u d i e s .t h el e c t i n
[ B rg/ml] wa5 i n c u b a t e dw i t hv a r y i n g
c o n c e n t r a t i o n so fs u g a r s
i n 0.5 rm f o r 2 h a t 25% and n i x e d w i t h an e q u a l
Excess o ft h el e c t i n[ 4
hemaggluv o l u m eo fs t a n d a r de r y t h r o c y t es u s p e n s i o n .
assay v o l w ] was u s e di nt h eh e m a g g l u t l n a t i o ni n h i t i n a t i o nu n i t si nf i n a l
b i t i o n assay t o ensure t h a t a g g l u t i n a t i o n is c o m p l e t ei nt h ea b s e n c eo f
a 2 hi n c u b a t i o nt h ea b s o r b a n c e
was measured a t6 2 0 nm.
i n h i b i t o r .A f t e r
P e r c e n t a g ei n h i b i t i o n W 8 5 C a l C u l a t L df r o mt h er a t i o
Of t h ea b s o r b a n c eo ft h e
t u b e sc o n t a i n i n g
a m i x t u r e of t h e l e c t i n and t h e i n h i b i t o r , t o t h o s e
cont a i n i n g cell s u s p e n s i o na l o n e .
The a m o u n to fi n h i b i t o r
giving the same
absorbance a6 t h a i of t h eC o n t r o l[ w i t h o u tl e c t i n ]
was t a k e n as t h e v a l u e f o r
1W.A i n h i b i t i o n .
Reduction o ft r i - N - a c e t y lc h i t o t r i o s e :
To d r i e dt r i - N - a c e t y lc h i t o t r i o s e
(10.0 WJ) i n PBS, sodiumbora ['HI
hydride(4.0
q. 100 m C i / m o l ) i n 5 0 p l
thereactionmixture
Was
Of 0.05 N sodium h y d r o x i d e s o l u t i o n Was addedand
i n c u b a t e d a t 50°C f o r 30 min. U n l a b e l l e d Sodium b o r o h y d r i d e (10.0 mg) was
Subsequentlyaddedandthereaction
war c o n t i n u e df o r
an a d d i t i o n a l 30 min.
The r e a c t i o n was t k n n i n a t e d by n e u t r a l i z a t i o n w i t h 0 . 7 5
N s u l p h u r i ca c i d .
The r e a c t i o nm i x t u r e was t h e nl o a d e d on a Biogal P-2 column e q u i l i b r a t e d i n
w a t e ra n da l i q u o t %o fe a c hf r a c t i o ne l u t e d
were counted on a s c i n t i l l a t i o n
Counter. The p e a kf r a c t i o n s
were p o o l e da n dt h ep u r i t y
was confirmed by t h i n
p y r i d ~ n e / e t h y l a c e t a t e / a c e L i c a c i d / w a t e r( 5 : 3 : 3 : 1 ) .
l a y e rc h r o m a t o g r a p h yu s i n g
E a u i i i b z i u md i a l v s l ~ :B i n d i n go fr a d i o a c t i v et r i - N - a c e t y lc h i t o t r i o a e
was
( 0 . 7m C i / m o l )o b t a i n e df r o mt h ep r e v i o u ss t e pt oL . a c u t a n u u l al e c t i n
A sample of 203 111 o f t h e l e c t i n
(13.12pM)
measured by e q u i l i b r i u m d i a l y s i s .
i n PBS-pME was i n t r o d u c e di n t o one compartment and a s o l u t i o n of 200 ) ~ 1of
v a r i o u sa m o u n t s Of adl lo active l i g a n d (0.012 t; 1.2 m
M
) was i n t r o d u c e d Into
t h e o t h e r colnpartment Of t h e d i a l y s i s c e l i .
The cells were s h a k e nf o r6 0h
a t 5OC andsamples were removedand
counted on a s c i n t i l l a t i o n c o u n t e r and
t h eb i n d i n gd a t aa n a l y s e d
aCcOPding t o S c a t c h a r d ( 6 6 ) .
FluorescenceEDeCtra:
The intrinsic f l u o r e s c e n c eo ft h eD r o t e i n
was r e c o r d e d
~~w i t h a Perkin-Elmer WF-44A s p e c t r o f l u o r i m e t e r using a 6 1 i t width of 5 m on
bothmonochromators.Thelectinin
PBSPMT Waa e x c i t e da t2 9 5
The
f l u o r i m e t r i c t i t r a t i o n s were perfarmed by a d d i t i o no fa i i u u o t sf r o m
a stock
sugar s o l u t i o n . When t h ef l u o r e S C e n C eo ft h el e c t i n
was m e a s u r e di nt h e
P r e s e n c e Of a s a c c h a r i d et w oc o n t r o lS d U t l o n sw i t hb u f f e r
alone and s a e c h b
r i d e alone were also meaiured. The f l u o r e s c e n c em e a s u r e m e n t s were p e r f o m e d
a t 20%.
~
~
~
~
NO.
CD SDeCtra:
The r p e c t r e were r e c o r d e dw i t h a Jasco 5-20 s p e c t r o p o l a r i m e t e r
i n a 1.0 m c e l l f o r t h e r e g i o n
200-250 nm and a 5.0 m c e l l f o r t h e r e g i o n
25-300 m. The d a t a are e x p r e s s e d as mean r e s i d u e e l l i p t i c l t i e s
(a) i n
deg cm2/dmol. t h e mean r e s i d u ew e i g h tb e i n gt a k e n
as 110. The p e r c e n t a g e of
s e c o n d a r y s t r u c t u r e of L . a c u t a n w l a a g g l u t i n i n was d e t e r m i n e d a c c o r d i n g t o
a p r o g r a mw r i t t e n
by
themethodofProvencherandGlockner(67)using
PrOvencher(68).
Titrations wereperformed by a d d i t i o no fa l i u u o t sf r o m
mean r e s i d u e e l l i p t i c i t y i n
s t o c k sugar s o l u t i o n and t h ee n h a n c e m e n ti nt h e
was m o n i t o r e d .T h ec u v e t t e s
were f i l l e d w i t h 1.0 m l of
t h ea r o m a t i cr e g i o n
i n a t h e m o s t a t i c( t
0.5OC)
p r o t e i ns o l u t i o n (35 phi) i n PRS-@ME andmounted
c o p p e rh o l d e r .
The t i t r a t i o n s were performed a t 15'. Zoo, 25' and 36%.
Resu_lrg
P u r i f i c a t i o n of h . a c u t a n w l aa g g l u t i n i n :
Most o ft h ea g g l u t i n a t i n ga c t i v i t y
p r e s e n t i n t h ep h l o e me x u d a t eo fL . a c u t a n e u l af r u i t s
was r e c o v e r e d i n t h e
6%
ammonium s u l f a t ef r a c t i o n .S u b s e q u e n tC u r i f l c a t i o n
was accomplished w i n g
soybean agglutinin glycopeptid~-Sepharoae-68 s o l m n ? as t h ec o r r e s p o n d i n g
g l y c o p e p t i d e was a very good i n h i b i t o r o f h e m a g g l u t l n a t i o n c a u s e d
by t h e
lectin.
The l e c t i n bound t ot h ea f f i n i t ya d s o r b a n t
was e l u t e d (111 a s i n g l e
peakwith0.1
M a c e t i ca c i d
and d i a l y s e d a g a i n s t PES-@ME t o remove a c e t i c
a c i d . Usually 2-22
mgs O f ' l e c t i n was obtainedfrom4.5
ml Of exudate
e x t r a c t e d f r m 5 kg f r u i t s .T h i s
amounts t o an overall recovery of 70-7VA
a specifichemagglutinationactivity
Of IC00
fromthecrudeextractwith
( T a b l e I). An e s t i m a t e of t h ea c t i v ep r o t e i ny i e l di n d i c a t e st h a t
8-1w of
t h et o t a lP r o t e i ni ne x u d a t eC o n s t i t u t e st h el e c t i n .
Downloaded from www.jbc.org by on June 16, 2008
SBA-G?
Fig.1:
structure
and composition
of g l y c o p e p t i d e s
and o l i g o s a c c h a r i d e su s e df o r
hemagglutination
s t u d i e s . The
f o l l o w i n ga b b r e v i a t i o n s are used:
5, sialic acid;
M mannose; 4.
g h a c t o s e ; G.
GlcNAc; N. a s p a r gine; SBA-GP,
s o y b e a na g g l u t i " i ng l y c o p e p t i d e ;
A Tetra-N-acetylchitotetraose-specificExudate Lectin
14626
Table I :
P u r i f i c a t i o no fi . e c u t a n w l aa g g l u t i n i n .
Table 111:
Amino a c i dc o m p o s i t i o no f& . a c u t a n o u l al e c t i n a .
h i n o acid
C ohim
ntent acid
Ala
9000x g
supernatant
'
5
6
(NH4)2SD4
fraction
Purified
lectin
t4e5
2'0.0
38.0
125.4
18.0
25.0
118
25.0
103.0
154
19.0
76.0
lox,
18.0
72.0
Of a c t i v i t y i s d e f i n e d as t h a t amount O f l e c t i n w h i c hg i v e s
X% a g g l u t i n a t i o n o f e r y t h r o c y t e s
i n t h e h e m a g g l u t i n a t i o n aaray.
a One U n i t
8
18
22
32
18
52
14
6
T
COnCentIation
Sugar
(e)
e
1
20
218
300
lox,
6.0
0.3
0.M7C
0.02
SBA-GP
0.m
Fet-GP
0.01
0.008
KF-3
0.W
ASF-CS
ob"
SBA-a5
LacNAc
MannobioscC
Mannotriore
GIcNAc
MeelrGlcNAc
MeeGleNAc
6CQ
750
750
857
0.037
>0.06'
160
>75
0.038
are @(!->4)
linked l.
tri, t e t r a and
pentar a c c h a r i d e s ' o i GlcNAC r e s p e s t i v e l y R
. e l a t i v ei n h i b i t o r yP m e r
is w l t h
r e s p e c tt o( G I C N A C ) ~ .
GalNAc (5CU mM), ManNAC (500 mM). L a c t o s e (400 mM).
C e l l o b i o s e (200 dl) M e l i b i o s e (400 &)
Maltase (400 DIM), Mannose (200 mM)
and ~ - ~ G ~ ~ N A E - ( I - ~ N ) ~ N Were
( ~ ~ W
"0:I ) i n h i b i t o r y a t c o n c e n t r a t i o n s
as
i n d i c a t e d i n p a r e n t h e s e s . *Higher c o n c e n t r a t i o n sC o u l dn o tb eu s e d
t h i s s a c c h a r i d e was l h i t i n g .
are a ( l - > 2 ) l i n k e dd i and t r i s a c c h a r i d e s o f
Mannobioseandmannctriose
mannose. r e s p e c t i v e l y .
a (GICNAC)~,~
a t y p i c a le q u i l i b r i u md i a l y o i se x p e r i E q u i l i b r i u md i a l y s i s :
The r e s u l t so f
ment are e x p r e s s e d In Fig.3.
The r e s u l t sp l o t t e da c c o r d i n gt oS c a t c h a r d
(66). on e x t r a p o l a t i o n a t o r d i n a t e y i e l d e ;
a value of 2.06, i n d i c a t i n gt w o
bindingsitesfortri-N-acetylchitOt2106eper
molecule Of L . a C u t a n q u l a l e c t i n .
was c a l c u l a t e d t a b e
2.75 x IO4 .'-M
T h eb i n d i n gc o n s t a n t
H e m a g q l u t i n a t i o na c t i v i t y :
All t h em o n o s a c c h a r i d e lt e s t e df a i l
t0 i n h i b i t
t h ea c t i v i t yo fL . a C u t a n Q u i aa g g l u t i n i n
even a t very h i g h c o n c e n t r a t i o n s
(Table N). The d i s a c c h a r i d e sC e l l o b i o 6 em a l t o s e . a e l i b i o s e
and l a c t o s e were
also ineffective.
The l e c t i n a c t i v i t y wa; i n h i b i t e d by @(I-M) l i n k e d
oligomersof
GlCNAc and t h e i r i n h i b i t o r y p o t e n c y i n c r e a s e d d r a m a t i c a l l y w i t h
increase i n c h a i nl e n g t h .N - l i n k e dg l y c o p e p t i d e s
of s o y b e a na g g l u t i n i n ,
1
F i g . 3E
. q u i l i b r i u md i a l y s i sd a t a
for tbhien d rioanfdg i o l a b e l l e d
t r i - N - a c e t v l c h i t o t r i o s et o
*u
1.0
2.0
r
4
D
9
I n t r i n s i cf l u o r e s c e n c es t u d i e s :
The f l u o r e s c e n c e emission spectrumof
L . a c u t a n O u l al e c t i n
given In Fig.4(A)shows
an emisrlon maximum a t 336 nm.
On a d d i t i o n t o t h e C h i t c - ~ l i g 0 l a c c h a ~ i d e s , t h e f l u o r e s c e n c e
is enhanced
maximum towards 332
F i g . 4 ( 8 ) shows
w i t h a b l u es h i f ti nt h ee m i s s i o n
t h ec h a n g e in f l u o r e s c e n c e i n t e n s i t y as a f u n c t i o no ft r i - N - a c e t y l c h i t o trioseconcentrationtogetherwith
(I re r e r e n t a t i v c g l d p h l c a l d e t e r m i n a t i o n
(Kay, a c c o r d i n ft z i t h e method O f
( i n s e t ) of t h ea s s o c i a t i o nc o n s t a n t
Chlpman e t a l . (69). y i e l d i n g a v a l u e of 1.26 X 10 M
The s t O i c h i m e t r y
Of l e c t i n - s u o a r b i n d i n q f o r t h e p r o t e i n w i t h
an e q u i v a l e n t w e i g h t o f
M,
24000 c o r r e s p o n d st o
1. The t h e r m o d y n a m i cp a r a m e t e r sf o rb i n d i n go f
various
s a c c h a r i d e sd e t e r m i n e d by t h i s m e t h o da l o n gw i t ht h ef l u o r e s c e n c ee m i s s i o n
them are l i s t e d i n Table V.
c h a r a c t e r i s t i c s of t h e l e c t i n s a t u r a t e d w i t h
NO.
.
a
b
Log Mr
r'(cm')
Fig.Z(A).
Polyacrylamide gel e l e c t r o p h o r e s i s Of & . a c u t a n g u l al e c t i n .
P r o t e i nl o a d e d on t h e gels was 40 pgs. ( a ) a t pH 8.3 i n 7.5% acrylamide
gels. ( b ) SDS gel eIeCtrophOreS16in
1 0 , gels. 2 ( 8 ) . Molecular
Weight
d e t e r m i n a t i o n o f L.acutanwla l e c t i n bySephadex
GlOO chromatography
(0
0 ) and SDS gel e l e c t r o p h o r e s i s (-)
The r e f e r e n c ep r o t e i n sa r e
A
r i b o n u c l e a s e (Mr 13mO),8,
chymotrVpsinogen'(23,000); C. ovalbumin (45.0001;
D 8 - l a c t o g l o b u l i n (18 403 i n el f i l t r a t i o n i t moves as a d h r 35,000):
E: bovine s e r m s l b u n d (6+,ooO?; F . human I G h e a v yc h a i n ( 5 0 . d ) .
The
p o s i t i o n s o f t h el e c t i n are i n d i c a t e d by ope: C i r c l e s . 2 ( C ) . S e d i m e n t a t i o n
A linear p l o t o f t h e l o g a r i t h
e q u i l i b r i m analysis of L . a c u t a n w l r l e c t i n .
sf t h e e q u i l i b r i m p r o t e i n a b s o r b a n c e
versus t h e Square o f t h e r a d i a l
is i n d i c a t i v e o f t h e s i r e h o m o g e n e i t y
Of
distancetothecentreoftherotor
the&.acutanQulalectin.
--
a Values u n a f f e c t e d by t h e p r e s e n c e
or abSence of p-mercaptoethano1.
s&
s e d i n e n t a t i o cno e f f i c i e n t .
R S t o k e s -r a d i u s .
D e n s i t y of 1.005 g m1-l and 1.15 u s e d f a t C a l c u l a t i o n s
were f o r p h o s p h a t e b u f f e r a n d b u f f e r c o n t a i n i n g
6 M GWClrespectively.
SE s e d i m e n t a t i o en q u i l i b r i m .
Downloaded from www.jbc.org by on June 16, 2008
h i n o a c i d and c a r b o h y d r a t e analyses: NO n e u t r a l sugar ( < 0 . 1 s ) couldbe
d e t e c t e d by p h e n o l - r u l p h u r i e a c i d method and no amino sugar was d e t e c t e d
d u r i n ga m i n o a c i d analysis. The p u r i f i e d l e c t i n d i d n o t b i n d t o Con *Sephamsc or t o ffiA-SephazOse.
T h e s ed a t at a k e nt o g e t h e ri n d i c a t et h a tt h i s
l e c t i ni sn o t
a glycoprotein.
The amino a c i dc o m p o s i t i o no ft h ea g g l u t i n i n
contains highamounts of a s p a r t i ca c i d
i sg i v e n in Tabla 111. T h i sp r o t e i n
a n dg l u t a m i ca c i dr e h i d u e sa n d / o rt h e i ra m i d e s
in a d d i t i o n t o l e u c i n e , V a l i n e
serine a n dg l y c i n eH
. ydroxyproline
i s a b s e n t . I t Contains two f r e eh a l f
hl s t o r a g et h ep r o t e i n
c y s t e i n e sw h i c h a m n o t C r i t i c a l f o r i t s a c t i v i t y .
pME a n d .t h e r e f o r e .
i t i s COnVenlent
t e n d s t o p r e c l p i t a t e in t h ea b s e n c eo f
t o use b u f f e r c o n t a i n i n g @ M E d u r i n g s t o r a g e .
~ e l a t i ~ e ~
inhibitory
power
1i n h i b i t i o n (mM)
-
~ 8 , ~
44
d
P u r i t v and nacrmolecular D r o p e r t i e s :P u r i f i e dL . s c u t a n a u l aa g g l u t i n i n
was
foundhmoganeous by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n SDS. a c i da n d
a l k a l i n e pH and a l s o by gel f i l t r a t i o n (Fig.2A).Thehomogeneityof
the
giving
l e c t i n was f u r t h e r c o n f i r m e d by s e d i m e n t a t i o n v e l o c i t y a n d B q u i l i b r i u m
e l p e r i m n t s . P o l y ~ c r y l . m i d e gel e l e c t r o p h o r e s i s i n t h ep r e s e n c e of SDS
On
y i e l d e d an M, Of 24ccO 2 lo60 ( F i g . 2 8 ) .G e l - f i l t r a t i o no ft h el e c t i n
Sephadex Glm. gave a s i n g l e a m e t r i c a l peakwith a m o I e c u I a r weightof
47000 + loo0 In Dresenceandabsenceof
BME (Fiq.28) a n d t h e e l u t i o n
prcf i l e m o n i t o r e d by absorbance a t 280 MI was c o i n c i d e n t w i t h t h e h e m a g g l u t i n a t i n ga c t i v i t y of t h el e c t i n .T h e s e
values remain u n a l t e r e dw i t h W E . The
was d e t e l r n i n e d t o b e
2.9 m from t h e
S t o k e s ' radius Of t h e n a t i v e p r o t e i n
gelchromatographyexperiments.
I n a e d l m e n t a t i o nv e l o c i t ye x p e r i m e n t s .t h e
a
valve of 4.06 S. She
i e c t l n g a v e a s l n q l es e d i m e n t i n gb o u n d a r yw i t h
value of
49500 2 1000 was Obtained using a p a r t i a ls p e c i f i c
volume
of
0.71 ml/g, a S t o k e s ' r a d i u s o f
2.9 m and a s e d i m e n t a t i o n c o e f f i c i e n t o f
4.06 5 .
For a molecular massof 4 8 m . a
of 4.06 S and
of 0.71 d g ,
t h e e s t i m a t e d f/fmin of l.M imply t h a t & . a c u t a n a u l a l e c t i n i s
a globular
protein
The s i z e homogeneity was f u r t h e rc o n f i r m e d
by s e d i m e n t a t i o n equii i b r i u m ' e x p e r f m a n t sw h e r e
a MI o f 48003 was obtained(Fig.2C).
On d i s s a R .., Of 2 . 1 5 5
C i a t l o n w i t n 6 M GWCl in t h e presence and aDsence Of B E . an S ZY."
w a s o b t a i n e di n d i c a t i n gt h a tt h el e c t i nd i s s o c i a t e di n t o
a 5peCleS which IS
h a l f i t s n a t i v e s i r s . A s i n g l e a m i n oa c i d glycine was found a t t h a m i n o
terminal end f u r t h e rs u b s t a n t i a t i n gt h eh o m o q e n e i t y
O f t h ep r o t e i n .T h e
macromolecular p r o p e r t i e s o f t h e l e c t i n
are l i r i t e d I n Table 11.
&,,
Content
(molhol)
Le"
30
A Tetra-N-acetylchitotetraose-specific
Exudate Lectin
T a b l e V:
14627
Fluorescence e m i s s i o nC h a r a c t e r i s t i c s .a S S o c i a t i o nc o n s t a n t sa n d
f r e e energy of a s s o c i a t i o n of s a c c h a r i d e s w i t h r . a o u t a n o u l a l e c t i n
a t 20%.
*
/
LO+-
x
2
I
1
Fi9.6:
v a n ' t Hoff p l footthr e
b i n d i n g of L.aeutanqula l e c t i n
t o tri-N-stetylchitotriose. K.
values f o rt h el e c t i n - s u g a r
i n t e r a c t i o n were determined by
4
4
a K, and t h e % e n h a n c e m e n t i n f l u o r e s c e n c e f o r t h e b i n d i n g o f
various
s a c c h a r i d e s *em C a l c u l a t e d a t t h e r e s p e c t i v e s h i f t e d f l u o r e s c e n c e
emission maxima f o r t h e s el i g a n d s .
Value6 i np a r e n t h e s e si n d i c a t e
standarddeviation.
Far W-CD rvect-:Analysis
of t h e CD s p e c t r u mo fr . a c u t a n q u l al e c t i n
gave 31% h e l i xC o n t e n t( F i g . % A ( a ) ) .
Near W-CG Spectrum:Near
W - 0 bands show an i n c r e a s e i n t h e
molar e l l i p t i The change i n
c i t i e s on t i t r a t i o nw i t hc h i t D - 0 l i g ~ 9 a e c h a r i d e s( F i g . 5 A ( b ) ) .
t h e m o l a r e l l i p t i c i t y as a f u n c t i o n of t h el i g a n dc o n c e n t r a t i o n( F i g . 5 8 )
a l l o w e dt h ed e t e r m i n a t i o n
of Ka a n dt h es t o i c h i o m e t r y
of t h e r e a c t i o n b y
p l o t t i n g log[ (ec $ ) / ( e ,
e = ) ]versus log[s], where eo, ec and ea are
sugar. m a a l l i p t i initialellipticity.ellipticityafteradditionofthe
C i t y value where all t h e b i n d i n g S i t e s
of t h e l e c t i n are s a t u r a t e d w i t h
s u g a r . r e s p e c t i v e l y .T h e
value of K, and t h es l o p eo ft h i sr e p r e s e n t a t i v e
p l o t Of t l t r a t i o n a t 2 0 % are 1.1 x lo4 M-'
and 1.0 r e s p e c t i v e l y( F i g . 5 C ) .
Value. Of Ka for v a r i o u s O l i g o s a c c h a r i d e s t h u s d e t e r m i n e d a t
several
t e m p e r a t u r e s were u t i l i z e d for e v a l u a t i n g A H by v a n ' t Hoff p l o t s (Fig.6)
and t h er e s u l t i n gt h e r m o d y n a m i cp a r a m e t e r 3
a m g i v e ni nT a b l e
VI.
-
-
-
a AH was o b t a i n e d frm v a n ' t Hoff p l o t s .
A S was c a l c u l a t e d using t h e
AH-Tns.
Values i no a r e n t h e s e si n d i c a t es t a n d a r dd e e q u a t i o nA G
viation.
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Fig.5. (A).
CD s p e c t r a of . . a s u t a n q u l al e c t i n .
( a ) The c o n c e n t r a t i o no f
f a r W r e g i o n (202-250 nm) was 0 02% in Pas c o n t a i n i n g
the lectin in the
2 nM d i t h i o t h r e i t o l and t h e near W r e g i o n (250-3W IM) was 0 9z ( b ) CD
e n h a n c e m e n tS p e c t r ao fL . a c u t a n a u l al e c t i nb yt = i - N - a c e t y l c h i i o t r i o s ei nt h e
near W region. Lwer curve r e p r e s e n t ss p e c t of the p r o t e i n alone and
of S a t u r e t i n g
t h eu p p e r Curve r e p r e s e n t s t h e f i n a l s p e c t r u m a f t e r a d d i t i o n
(8)
Curve of t h e m o l a r e l l i p t i c o n c e n t r a t i o nostfr i - N - a c e t y l c h i t o t r i o s e
c i t ye n h a n c e m e n to f& . a c u t a n a u l al e c t i n
b; tli-ii-aCetylghitotriose+
IS1