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Analytical and semi-preparative high-performance liquid chromatography of oligosaccharides obtained by hydrazinolysis of hen ovomucoid
Jmunzf of Chrmmograph~, Elsewer Sctentdic Pubhshmg CHRO,M_
249 ( 1982) 199-204 Company, Amsterdam
-
Prmted
m The Netherlands
15.156
Noze
Analytical
graphy coid JOSC
PAZ
BERNARD
and semi-preparatiwe
of oligosaccharides
PARENTE,
GERARD
high-performance
obtained
STRECKER
by hydrazinolysis
YVES
LEROY,
liquid chromato-
JEAN
of hen owomu-
MONTREUIL
and
FOURNET-
Inbora~oue de Chutup Bwlog~que de I’Umrersrre a&sSrrpnces er ~echmqws de L.dk ler Loboraro~re ~ssocre m C N R S No 217,596X5 - Yllleneule dAscq Ceder (France) (Fwst rece~wd June 2nd,
1982. reused
manuscnpt
reccwed
June 30th.
1982)
Currently, monosaccharides and ohgosacchandes are satrsfactonly separated by paper, ion-exchange, thm-layer and gel Ntratton chromatography, however the methods are t~e-consummg Hugh-performance hqurd chromatography (HPLC) using a column of porous mrcropartrcles denvatrzed wrth ammo groups allows a rapid separation of sugars I5 Since 1980, the HPLC on bonded pnmary amme packings of sugars denved from glycoprotem glycans have been descnhed fractronatton of ohgosaccharides and glycopeptrdes excreted m the urme of patients wrth lysosomial disease@; separations of ohgosacchandes obtained by drgestton wtth endoglycosrdase H’ or released by &ehmmatron of 0-glycosyiprotem* g, ohgosacchandes denved from dohchol-linked ohgosacchande mtermedtates’” Finally, clear separations of staloglycopeptrdes or ohgosacchandes have been obtamed m less than 1 h by HPLC on a Micropak AX-10 ron-exchange column” The present report shows that bonded pnmary amme packings are effecttve m the raprd separation of the mixture of neutral glycans hberated by hydrazmolysts of a hen ovomucord neutral glycopeptrde MATERIALS
AND
METHODS
Cl” coprotems, gl’copeptldes
and ohgosaccharIdes
Ovomucotd was prepared accordmg to Fredencq and Deutschl’ The aaaloglycopeptrdefl was Isolated after pronase hydrolysis of ovomucoid accordmg to ~Monsrgny et a1.13. Oligosacchandes were released from astaloglycopeptrde j? by hydrazrnolysts as prevtously descnbed’4 The resultmg ohgosaccharides were N-reacetylated accordmg to Readmg et al I5 and reduced wtth NaBH, Liqmd chromatography
on pnmary
amne
bonded s&a
Analysis were can-ted out with a Spectra Physics Model 700 hquxd chromatograph, equipped wrth an UV 8400 vanable wavelength detector connected to a 4100 compuhng Ir&gratOr. HPLC was performed on a 5-w Ammo AS-SA column (0.4 x 25 cm, Chromatem 33; Touzart et Mat&on). A I-mg amount of oligosaccharides drssolved m 10 0021-9673/82~0W0-0WWS02.~5
Q
1982 EIsewer
Screnbfic
Pubhshmg
Company
200
NOTES
pL1of lstrlled water was injected mto the column; for preparative chromatography, 3.5 mg of oligosacchandes dissolved in 20 4 of acetonitriie-water (50-50) were injected_ The column was eqmhbrated with the mitial soivent (acetontnle-water, 6.5:35)_After the injection, a linear gradient to acetomtnIe-water (6OAO) was applied for 30 min followed by isocratic cond&ons during 30 min and then a linear gradlent to acetonitnle-water (50.50) for 30 min. The now-rate was 1 mlfmin. The oligosaccharides were detected at 200 nm under-tie following conditions- sensitivity of detec-
&?L I- ~ys= ofohghdcs 5% ~chromatern
(A) of ohgosacchandes1,7, !I and I4 obtainedby sum-prepazhx chromatography(B) from the h~drapnoljrusof hen ovomucotdneutralglycopcptide/3on5-p Amino As 33. Towart ct Mabgnon) For chromatograpiuccondition%see MatenaI and Methods.
NOTES
201
tor, 0.35; integrator attenuation, 4, for analytical chromatography; sensltlvlty of detector, 0 32, integrator attenuatxon, 50, for semi-preparative chromato_gaphy. Molar
conrposillon of ohgosaccharrdes The molar composrtron of ohgosacchandes
was determmed by gas-hqurd chromatography (GLC) of trtfluoroacetylated methylglycosrdes accordmg to Zanetta et aL16. Thm-iajer
chromatography
(TLC)
of ohgosacchartdes
TLC of oligosacchandes was performed on sthca gel plates (pre-coated sthca gel 60, Merck) using ethanol-n-butanol-pyridine-acetic actd-water (100.10 10.3 30 v/v/v/v/v)” dunng 7 h. Oligosacchandes were revealed with an orcmol-sulphunc acrd reagent’* RESULTS AND DISCLJSSION
The separatron of a mrvture of reduced ohgosacchandes obtamed by hydraztnolysts from hen ovomucotd ISshown m Frg I The effecttve separation of seventeen fractrons was obtamed wtthm 90 mm on an Ammo AS-SA column (Frg. 1B) and semt-preparatrve chromatography on the same column allows one to obtain pure fractions 1, 7, ! 1 and 14 (Fig. IA) The results of the semi-preparative cbromatography of 4.2 mg of ovomucotd-denved ohgosacchandes are gtven m Table I.
TABLE 1 WEIGHTS OF FRACTIONS OBTAINED BY SEMI-PREPARATIVE CHROMATOGRAPHY OF 4 2 mg OF OLIGOSACCHARIDES LIBERATED BY HYDRAZINOLYSIS FROM OVOMUCOID NEUTRAL GLYCOPEPTIDE /3 Fracrron number F-O F-I F-2 F-3 F-4 F-S F-6 F-7 F-8 F-9 F-IO F-11 F-12 F-13 F-14 F-15 F-16 F-17 Recovered
Werghr (,ug) 3124 4s 363 95 7 1694 73 7 265 5104 1463 2838 4ca4 6446 186 242 284 484 36 3 83 7 3862
NOTES
202
The use of a 0 4 x 25 cm column filled with srlica gel modtied by organic amines provides quantitative recovery (92 “4) of the ohgosacchzridcs wrthout loss of resolution_ Each fraction was analysed by TLC (Fig. 2) and the carbohydrate composition was deter-mu& by GLC (Table II). Four fractions (1, 7, 11 and 14) are homogeneous on the basis of HPLC and TLC and of the monosaccharide molar composition_ The latter is characterized by the absence of the presence in low amounts of galactose and by the relatrve high content of N-acetylglucosamme. Of interest is the ratio (N-acetylglucosamine + N-acetylglucosanumtol)/mannose, I e , (GlcNAc + GlcNAc-ol)/Man, the value of which is related to the number of Nacetylglucosamine branches on the mannose residues_The linuting values are 1.35 and 2.7 in the case of classical biantennary structures such as human serum transferTin” ,md of pentaantennary structuressuch as turtle-dove ovomucoid”, respectively. CONCLUSIONS
HPLC on bonded primary amine packings gives excellent fractionahon cf a
Fig- 2. TLC on sbca gel plates (Merck) of seventeen frations obtamed by seou-preparabve chromatography of obgosacchanda from the hydtazmoIyss of hen or omucotd neutral glycopephde Solvent: ethanoin-butaool-pyndm-cetlc acid-war(100 IO_10330 \j\jvjvjv) Development time- 7 b Ohgosacchanda rexaXed aitb orcinokulphtuic aad reagent
NOTES TABLE
203 Ii
CARBOHYDRATE COMPOSITION OF FRACTIONS OBTAINED BY SEMI-PREPARATIVE CHROIMATOGRAPHY OF OLIGOSACCHARIDES LIBERATED BY HYDRAZINOLYSIS OF HEN OVOiMUCOID NEUTRAL GLYCOPEPTIDES B Fracttons
Molar rano* Gal
1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17
Man
0 0 0 0 0 0 0 041 044 059 019 072 103 107 152 15 185
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
06
3
z
GIcNAc i- GkNAc-ol GkNAc
Man
GlcN 4c-of
13 2 94 34 3 63 34 39 464 502 5 38 463 65 570 599 68 69 6 11 68
094 081 0 50 0 66 02 060 094 061 065 064 101 0 57 0 30 I 07 071 0 57 0 84
0 74 I 25 13 143 12 1.5 I 86 1 87 2 01 1 75 2x5 2-09 209 2 62 2 53 2 26 2 54
Natae
glyopeptidep
6
2
* The M~IO of mannose (Mm) was taken JS 3 Gal = Galactose. GlcNAc GlcNAc-01 = i*c-acetylglucos~mltol
= Y-acet&lucosamme.
new class of glycans constltutm g a complex mixture of about twenty reduced neutral ohgosacchandes liberated by hydrazmolysis from hen ovomucold- These glycans are m general N-acetylglucosamme-nch and highly branched Our results Illustrate agam the usefulness of HPLC as a powerful tool to investigate the various compounds encountered m the fieId of glycoprotems- glycans of the “N-acetyl-lactosaminx type”’ ‘I, of the “ohgomannosldlc type”‘, of the “mucm type”’ ’ and hpld mtermedlate-denved ohgosacchandeslo, dohchyl pyrophosphoryl ohgosacchandes” and peracetylated mono- and ohgosacchandes” The results obtained demonstrate the htgh mlcroheterogenelty of hen ovomucold, whxh was not revealed by our first mvestlgatlonsz3 The pnmary structure of the numerous giycans Isolated IS now under mvestlgatzon m order to understand the s~gmficance of their amazmg mlcroheterogenelty better and define the charactenstlcs of the new class of glycans ACKhOWLEDGEMENTS
This work was supported by the Centre National de la Recherche Sclentlfique (LA No 217) and the Wlegatlon G&&ale a la Recherche Sclentdique et Technique (79 7 0669)
NGTES
204
The authors are pateful to B_ Mahieu, G. Tine1 and J_ Celen for their skilful assistance and to the Sp&ra Physics Society for their constant cooperation and help dturing the work. REFERENCES I 2 3 4 5 6 7 S 9 IO 11 12 13 14 15 16 17 18 19 20 21 22 23
J. C_ tideu and C. L._ Lawhead. J Chromrrogr , 105 (1973 125-133 J K Pzhner? A&_ Letl~ 8 (1975) 215 L C_ Conrad and J K. Palmer. Food Technol, Oct. (1976) S4 R B Meagher S J and A_ Furs, J Chromutogr. 117 (1976) 211-215 K_ Ameanliller. J. Chronrmogr, 156 (1978) 354 N_ hi_ K_ Ng kmg Km and L_ S_ Wolf&. Anal. BlocEem . 102 (1980) 213-219 S J hlelbs and J U Elaenz~ger~&mI_ Bzochem, I14 (1981) 276-280 A Boersum, G_ Lambhn, P Degand and P Roussel, Corboh>d Res. 94 (1981) C7-C9. M 1- E Bergh. P. Koppeu and D H_ mn den fiJnden. Curboh,d_ Rer .94 (1981) 225-719 S_ J_ Turco, AML Blochem ) 1 IS (1981) 27S-283 J U_ Baennger and M Natowia, Anal Btochenz , 112 (1981) 357-361 E Fredeticq and H F_ Deutsch, L BxoI. Chem ~ 181 (1949) 499_ hl_ Mormgny. A_ Adam-Chosson and J Montreud, Bull Sot Chzm Brol ,50 (1968) 857-874 B Bayard and B. Foumet, Curboh_wL Res, 46 (1975) 75-86. C_ L_ Readmg E_ Penhoet and C. BalIott. J Emi Chem ,253 (1978) X00-5612 J P_ Zanetta_ W_ C_ Brechenndge and G Vincendon, J_ Chromrogr ,69 (1972) 291-304 B Bayard. J P_ Kerckaert. D Roux and G Streeker, ProrIdes St01 F7mc!s, Proc- Colfoq , 27 (1979) 153-156 R Humbel and M_ Collaen Chn_ Ck Acta, 60 (1975) 143-145 G- Splk. B- Ba>ard. B_ Fournet, G_ Strecker. S Bouquelet and J Montreud, FEES Lert ,50 (1975) 23~999_ Cb_ Fran~sGerard, J Brocteur, A Andre, C Gerday, A Pm-ee-Cretel, J_ Montreuil and G Spik, Blood Tram Invnunohaeamar ,23 (1980) 579-587 G. B_ \Vells, S J_ Turco. B A Hanson and R. L Lester, Anal Biochcnr , I10 (1981) 397406 G B_ Wells and R L. Lester, Anoi Bmchem ,97 (1979) 184-190 B_ &yard and J_ ~Montretul, ricres & ColIoque Intentationai No 111 du C_N R S sur les Gi~coconJU~I&S.~dIeneure dciscq, June Z&27_ 1973. Centre National de la Recherche Saentdique, Pans, 1974, pp 209-218
249 ( 1982) 199-204 Company, Amsterdam
-
Prmted
m The Netherlands
15.156
Noze
Analytical
graphy coid JOSC
PAZ
BERNARD
and semi-preparatiwe
of oligosaccharides
PARENTE,
GERARD
high-performance
obtained
STRECKER
by hydrazinolysis
YVES
LEROY,
liquid chromato-
JEAN
of hen owomu-
MONTREUIL
and
FOURNET-
Inbora~oue de Chutup Bwlog~que de I’Umrersrre a&sSrrpnces er ~echmqws de L.dk ler Loboraro~re ~ssocre m C N R S No 217,596X5 - Yllleneule dAscq Ceder (France) (Fwst rece~wd June 2nd,
1982. reused
manuscnpt
reccwed
June 30th.
1982)
Currently, monosaccharides and ohgosacchandes are satrsfactonly separated by paper, ion-exchange, thm-layer and gel Ntratton chromatography, however the methods are t~e-consummg Hugh-performance hqurd chromatography (HPLC) using a column of porous mrcropartrcles denvatrzed wrth ammo groups allows a rapid separation of sugars I5 Since 1980, the HPLC on bonded pnmary amme packings of sugars denved from glycoprotem glycans have been descnhed fractronatton of ohgosaccharides and glycopeptrdes excreted m the urme of patients wrth lysosomial disease@; separations of ohgosacchandes obtained by drgestton wtth endoglycosrdase H’ or released by &ehmmatron of 0-glycosyiprotem* g, ohgosacchandes denved from dohchol-linked ohgosacchande mtermedtates’” Finally, clear separations of staloglycopeptrdes or ohgosacchandes have been obtamed m less than 1 h by HPLC on a Micropak AX-10 ron-exchange column” The present report shows that bonded pnmary amme packings are effecttve m the raprd separation of the mixture of neutral glycans hberated by hydrazmolysts of a hen ovomucord neutral glycopeptrde MATERIALS
AND
METHODS
Cl” coprotems, gl’copeptldes
and ohgosaccharIdes
Ovomucotd was prepared accordmg to Fredencq and Deutschl’ The aaaloglycopeptrdefl was Isolated after pronase hydrolysis of ovomucoid accordmg to ~Monsrgny et a1.13. Oligosacchandes were released from astaloglycopeptrde j? by hydrazrnolysts as prevtously descnbed’4 The resultmg ohgosaccharides were N-reacetylated accordmg to Readmg et al I5 and reduced wtth NaBH, Liqmd chromatography
on pnmary
amne
bonded s&a
Analysis were can-ted out with a Spectra Physics Model 700 hquxd chromatograph, equipped wrth an UV 8400 vanable wavelength detector connected to a 4100 compuhng Ir&gratOr. HPLC was performed on a 5-w Ammo AS-SA column (0.4 x 25 cm, Chromatem 33; Touzart et Mat&on). A I-mg amount of oligosaccharides drssolved m 10 0021-9673/82~0W0-0WWS02.~5
Q
1982 EIsewer
Screnbfic
Pubhshmg
Company
200
NOTES
pL1of lstrlled water was injected mto the column; for preparative chromatography, 3.5 mg of oligosacchandes dissolved in 20 4 of acetonitriie-water (50-50) were injected_ The column was eqmhbrated with the mitial soivent (acetontnle-water, 6.5:35)_After the injection, a linear gradient to acetomtnIe-water (6OAO) was applied for 30 min followed by isocratic cond&ons during 30 min and then a linear gradlent to acetonitnle-water (50.50) for 30 min. The now-rate was 1 mlfmin. The oligosaccharides were detected at 200 nm under-tie following conditions- sensitivity of detec-
&?L I- ~ys= ofohghdcs 5% ~chromatern
(A) of ohgosacchandes1,7, !I and I4 obtainedby sum-prepazhx chromatography(B) from the h~drapnoljrusof hen ovomucotdneutralglycopcptide/3on5-p Amino As 33. Towart ct Mabgnon) For chromatograpiuccondition%see MatenaI and Methods.
NOTES
201
tor, 0.35; integrator attenuation, 4, for analytical chromatography; sensltlvlty of detector, 0 32, integrator attenuatxon, 50, for semi-preparative chromato_gaphy. Molar
conrposillon of ohgosaccharrdes The molar composrtron of ohgosacchandes
was determmed by gas-hqurd chromatography (GLC) of trtfluoroacetylated methylglycosrdes accordmg to Zanetta et aL16. Thm-iajer
chromatography
(TLC)
of ohgosacchartdes
TLC of oligosacchandes was performed on sthca gel plates (pre-coated sthca gel 60, Merck) using ethanol-n-butanol-pyridine-acetic actd-water (100.10 10.3 30 v/v/v/v/v)” dunng 7 h. Oligosacchandes were revealed with an orcmol-sulphunc acrd reagent’* RESULTS AND DISCLJSSION
The separatron of a mrvture of reduced ohgosacchandes obtamed by hydraztnolysts from hen ovomucotd ISshown m Frg I The effecttve separation of seventeen fractrons was obtamed wtthm 90 mm on an Ammo AS-SA column (Frg. 1B) and semt-preparatrve chromatography on the same column allows one to obtain pure fractions 1, 7, ! 1 and 14 (Fig. IA) The results of the semi-preparative cbromatography of 4.2 mg of ovomucotd-denved ohgosacchandes are gtven m Table I.
TABLE 1 WEIGHTS OF FRACTIONS OBTAINED BY SEMI-PREPARATIVE CHROMATOGRAPHY OF 4 2 mg OF OLIGOSACCHARIDES LIBERATED BY HYDRAZINOLYSIS FROM OVOMUCOID NEUTRAL GLYCOPEPTIDE /3 Fracrron number F-O F-I F-2 F-3 F-4 F-S F-6 F-7 F-8 F-9 F-IO F-11 F-12 F-13 F-14 F-15 F-16 F-17 Recovered
Werghr (,ug) 3124 4s 363 95 7 1694 73 7 265 5104 1463 2838 4ca4 6446 186 242 284 484 36 3 83 7 3862
NOTES
202
The use of a 0 4 x 25 cm column filled with srlica gel modtied by organic amines provides quantitative recovery (92 “4) of the ohgosacchzridcs wrthout loss of resolution_ Each fraction was analysed by TLC (Fig. 2) and the carbohydrate composition was deter-mu& by GLC (Table II). Four fractions (1, 7, 11 and 14) are homogeneous on the basis of HPLC and TLC and of the monosaccharide molar composition_ The latter is characterized by the absence of the presence in low amounts of galactose and by the relatrve high content of N-acetylglucosamme. Of interest is the ratio (N-acetylglucosamine + N-acetylglucosanumtol)/mannose, I e , (GlcNAc + GlcNAc-ol)/Man, the value of which is related to the number of Nacetylglucosamine branches on the mannose residues_The linuting values are 1.35 and 2.7 in the case of classical biantennary structures such as human serum transferTin” ,md of pentaantennary structuressuch as turtle-dove ovomucoid”, respectively. CONCLUSIONS
HPLC on bonded primary amine packings gives excellent fractionahon cf a
Fig- 2. TLC on sbca gel plates (Merck) of seventeen frations obtamed by seou-preparabve chromatography of obgosacchanda from the hydtazmoIyss of hen or omucotd neutral glycopephde Solvent: ethanoin-butaool-pyndm-cetlc acid-war(100 IO_10330 \j\jvjvjv) Development time- 7 b Ohgosacchanda rexaXed aitb orcinokulphtuic aad reagent
NOTES TABLE
203 Ii
CARBOHYDRATE COMPOSITION OF FRACTIONS OBTAINED BY SEMI-PREPARATIVE CHROIMATOGRAPHY OF OLIGOSACCHARIDES LIBERATED BY HYDRAZINOLYSIS OF HEN OVOiMUCOID NEUTRAL GLYCOPEPTIDES B Fracttons
Molar rano* Gal
1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17
Man
0 0 0 0 0 0 0 041 044 059 019 072 103 107 152 15 185
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
06
3
z
GIcNAc i- GkNAc-ol GkNAc
Man
GlcN 4c-of
13 2 94 34 3 63 34 39 464 502 5 38 463 65 570 599 68 69 6 11 68
094 081 0 50 0 66 02 060 094 061 065 064 101 0 57 0 30 I 07 071 0 57 0 84
0 74 I 25 13 143 12 1.5 I 86 1 87 2 01 1 75 2x5 2-09 209 2 62 2 53 2 26 2 54
Natae
glyopeptidep
6
2
* The M~IO of mannose (Mm) was taken JS 3 Gal = Galactose. GlcNAc GlcNAc-01 = i*c-acetylglucos~mltol
= Y-acet&lucosamme.
new class of glycans constltutm g a complex mixture of about twenty reduced neutral ohgosacchandes liberated by hydrazmolysis from hen ovomucold- These glycans are m general N-acetylglucosamme-nch and highly branched Our results Illustrate agam the usefulness of HPLC as a powerful tool to investigate the various compounds encountered m the fieId of glycoprotems- glycans of the “N-acetyl-lactosaminx type”’ ‘I, of the “ohgomannosldlc type”‘, of the “mucm type”’ ’ and hpld mtermedlate-denved ohgosacchandeslo, dohchyl pyrophosphoryl ohgosacchandes” and peracetylated mono- and ohgosacchandes” The results obtained demonstrate the htgh mlcroheterogenelty of hen ovomucold, whxh was not revealed by our first mvestlgatlonsz3 The pnmary structure of the numerous giycans Isolated IS now under mvestlgatzon m order to understand the s~gmficance of their amazmg mlcroheterogenelty better and define the charactenstlcs of the new class of glycans ACKhOWLEDGEMENTS
This work was supported by the Centre National de la Recherche Sclentlfique (LA No 217) and the Wlegatlon G&&ale a la Recherche Sclentdique et Technique (79 7 0669)
NGTES
204
The authors are pateful to B_ Mahieu, G. Tine1 and J_ Celen for their skilful assistance and to the Sp&ra Physics Society for their constant cooperation and help dturing the work. REFERENCES I 2 3 4 5 6 7 S 9 IO 11 12 13 14 15 16 17 18 19 20 21 22 23
J. C_ tideu and C. L._ Lawhead. J Chromrrogr , 105 (1973 125-133 J K Pzhner? A&_ Letl~ 8 (1975) 215 L C_ Conrad and J K. Palmer. Food Technol, Oct. (1976) S4 R B Meagher S J and A_ Furs, J Chromutogr. 117 (1976) 211-215 K_ Ameanliller. J. Chronrmogr, 156 (1978) 354 N_ hi_ K_ Ng kmg Km and L_ S_ Wolf&. Anal. BlocEem . 102 (1980) 213-219 S J hlelbs and J U Elaenz~ger~&mI_ Bzochem, I14 (1981) 276-280 A Boersum, G_ Lambhn, P Degand and P Roussel, Corboh>d Res. 94 (1981) C7-C9. M 1- E Bergh. P. Koppeu and D H_ mn den fiJnden. Curboh,d_ Rer .94 (1981) 225-719 S_ J_ Turco, AML Blochem ) 1 IS (1981) 27S-283 J U_ Baennger and M Natowia, Anal Btochenz , 112 (1981) 357-361 E Fredeticq and H F_ Deutsch, L BxoI. Chem ~ 181 (1949) 499_ hl_ Mormgny. A_ Adam-Chosson and J Montreud, Bull Sot Chzm Brol ,50 (1968) 857-874 B Bayard and B. Foumet, Curboh_wL Res, 46 (1975) 75-86. C_ L_ Readmg E_ Penhoet and C. BalIott. J Emi Chem ,253 (1978) X00-5612 J P_ Zanetta_ W_ C_ Brechenndge and G Vincendon, J_ Chromrogr ,69 (1972) 291-304 B Bayard. J P_ Kerckaert. D Roux and G Streeker, ProrIdes St01 F7mc!s, Proc- Colfoq , 27 (1979) 153-156 R Humbel and M_ Collaen Chn_ Ck Acta, 60 (1975) 143-145 G- Splk. B- Ba>ard. B_ Fournet, G_ Strecker. S Bouquelet and J Montreud, FEES Lert ,50 (1975) 23~999_ Cb_ Fran~sGerard, J Brocteur, A Andre, C Gerday, A Pm-ee-Cretel, J_ Montreuil and G Spik, Blood Tram Invnunohaeamar ,23 (1980) 579-587 G. B_ \Vells, S J_ Turco. B A Hanson and R. L Lester, Anal Biochcnr , I10 (1981) 397406 G B_ Wells and R L. Lester, Anoi Bmchem ,97 (1979) 184-190 B_ &yard and J_ ~Montretul, ricres & ColIoque Intentationai No 111 du C_N R S sur les Gi~coconJU~I&S.~dIeneure dciscq, June Z&27_ 1973. Centre National de la Recherche Saentdique, Pans, 1974, pp 209-218