Micromethod for fractionation of acid mucopolysaccharides

$Page 1: Micromethod for fractionation of acid mucopolysaccharides$
Clin. Biochem. (9, (1) 9-15 (1976)

Micromethod for Fractionation of Acid Mucopolysaccharides

N E L L Y B L U M E N K R A N T Z a n d G U S T A V A S B O E - H A N S E N

U n i v e r s i t y o f C o p e n h a g e n , D e p a r t m e n t o f D e r m a t o l o g y ( w i t h C o n n e c t i v e T i s s u e R e s e a r c h L a b o r a t o r i e s ) , R i g s h o s p i t a l , B l e g ~ l a m s v e j 9, 2100 C o p e n h a g e n ~ , D e n m a r k

( A c c e p t e d A u g u s t 20, 1975)

CLBIA, 9, (1) 9-15 (1976) Clin. Biochem.

Blumenkrantz, Nelly and Asboe-Hansen, Gustav

University of Copenhagen, Department of Der- matology (with Connective Tissue Research Lab- oratories), Rigshospital, Blegda.msvej 9, °.100, Copenhagen ~ Denmark.

MICROMETHOD FOR FRACTIONATION OF ACID MUCOPOLYSACCHARIDES

A new micromethod for fractionation of acid mucopolysaccharides based upon the use of different concentrations of HCI to separate the complex of CPC with non-sulphated, monosulphated and polysulphated acid mucopolysaccharides (glyco- saminoglycans) is presented. The method utilizes the different binding of the anionic macromolecules to the cationic compound cetyl pyridinium chloride. The method is simple and reproducible. The use of HC1 as eluent allows the exclusion of some steps required when salts are used for elution. Hexuronic acids are determined on the eluents.

SINCE SCHILLER ET AL"' r epo r t ed the i r method for f r ac t i ona t i on of acid mucopolysacchar ides (syn. acid g lycosaminog lycans ) , severa l p rocedures for micro and macrosca le f r a c t i ona t i on of these an ionic macromolecules have been proposed ' : ' ~). They a re based on the f o r m a t i o n of a complex w i th ce ty lpy r id in ium chlor ide (CPC) , a q u a t e r n a r y ammonium compound. Schi l le r et al ~) used inc reas ing concen t ra t ions of NaC1 to s e p a r a t e hya lu ron ic acid f rom monosu lpha ted and polysu lpha ted mucopolysacchar ides (i.e. chon- d r o i t i n su lpha tes and hepar in , r e spec t ive ly ) . The method was based on the f ac t t h a t the counter - ion b i nd ing capac i ty of su lpha te g roups is s t r o n g e r t han t h a t of ca rboxy la t e "~. In o the r p rocedures ~2. 3~ the p r ec ip i t a t i on w i th CPC was p e r f o r m e d on a cellulose column and f r a e t i o n a t e d e lu t ion of the A M P s obta ined wi th a se r ies of more or less empi r i ca l ly chosen solvents~ffi'sL T h e r a f t e r , u ron ic ac ids (UA, ~'j) or hexosamines (g lucosamine, ga l ae tosamine ; ~ were de t e rmined on the f rac t ions . A new micromethod fo r spec i f ic f r a c t i ona t i on of acid mucopolysacchar ides is r epor ted below. The method is based o'n the fo rma- tion of a complex between the A M P s and CPC and the d i f f e r e n t s e n s i t i v i t y of these complexes to va r ious concen t ra t ions of HCl.

MATERIALS AND METHODS

Acid mucopolysaccharides. Hyaluronic acid (HA), under- and oversulphated chondroitin sulphate (CS), chondroitin sulphates A, B and C (CSA, CSB, CSC; syn. chondroitin-4-

sulphate, dermatan sulphate, chondroitin-6-sulphate), heparitin monosulphate (HMS; syn. heparan sulphate), kerato-sulphate (KS; syn. keratan sulphate) 1 and 2 and heparin (Hep) were presents of Drs. Martin B. Mathews and John Cifonelli, University of Chicago, Ill.

Glucuronic acid was a gift from Martin B. Mathews.

Galactose p.a. was a product of E. Merck A. G., Darm- stadt.

Hyaluronidase (Leo, Hiilsingborg, Sweden) was prepared from bovine testes. The activity was 15.000 - - 20.000 U/mg.

Chondroitinases. Chondroitinase A, B, C. (Ch-ase A, B, C) al:d chondroitinase A, C (Ch-ase A, C) were purchased from Miles-Seravac Ltd., Monegrow Green Holyport, Maiden-head, Berkshire, England. Both were products of Sukagaku, Kogyo Co., Ltd., Tokyo, Japan..

Microcolumns. Disposable plastic micropipettes (Dansk Lahoratorieudstyr, Copenhagen, Denmark).

Celite 545, 30-80 mesh (Bie & Berntsen, Copenhagen).

Pal,ain (Worthington Biochemical Corp~,ration, Freehold, New Jersey), a twice crystallized suspension in 0.05 M sodium acetate pH 4.5, 39 mg/ml. Activity 15.8 U/rag.

EDTA. Ethylene diamine te t ra-acetate disodium salt p.a. (E. Merck A. G., Darmstadt) .

Cysteine HCI. (E. Merck A. G., Darmstadt) . m-Hydroxydiphenyl (analytical grade) was obtained from K & K Laboratories, Inc., Plainview, N.Y., or Eastman Kodak Co., Rochester, N.Y.

Anthrone p.a. was a product of E. Merck A. G., Darm- stadt.

Sulphuric acid. Concentrated sulphuric acid. specific gravity 1.84; analytical grade (E. Merck A. G., Darm- s tadt) .

Sodium tetraborate p a. (Riedcl de Hahn A. G., Seelze, Hannover).

Sodium hydroxide p.a. (E. Merck A.G., Darmstadt) .

Hydrochloric acid, analytical grade, specific gravi ty 1.19 (E. Merck. A.G., Darmstadt) .

Cetylpyridinium chloride p.a. (E. Merck A. G., Darm- stadt) ,

Reagents 1) Eluents. The solutions of HC1 in distilled water were adjusted to normalities of 0.005; 0.5; 2.0; 3.0 and 6.0. 2) 10% CPC in,disti l led water 3) 0.02 N NaCl in a 10% aqueous solution of CPC. 4) A 1% CPC solution was prepared by dilution of the 10% aqueous solution. 5) A 0.5% NaOH in water. 6) m-Hydroxydiphenyl, a 0.15% solution in 0.5% NaOH. The reagent was kept in the ref r igera tor covered by aluminum foil (5). 7) H~.SOdsodi'nm tetraborate, a 0:0125 M solution of te t raborate in concentrated sulvhuric acid '~'. 8) Standard glueuronic acid. Aqueous solutions con- raining 1, 2 and 4 .~g glucoronic acid per 0.2 ml. 9) Standard galaetos6. A solution of galactose containing 12.5 ~tg per 0.25 ml water.

10 B L U M E N K R A N T Z and A S B O E - H A N S E N

10) Preparation of celitc colu~n. Celite was suspended in 10% CPC, and 1 ml of the suspension applied to a disposable plast ic column of 3 cm with glass wool a t the bottom. The column packed wi th 1-3 cm celite was eluted with 0.02 N NaCl in 10% CPC to ensure sa tu ra t ion with the q u a t e r n a r y ammonium compound. The columns were p repa red immediate ly before use. D i f f e r e n t lengths of the celite columns were used according to the A M P content of the sample submit ted to f rac t ionat ion , i.e. 1 cm column was used fo r an A M P content up to 20 ~g; 2 cm for 20-40 i~g, and 3 cm for over 40 ~g A M P per 50 ~l aqueous solution of the sample.

Tr is -HCl buf fe r pH 7.3. Stock solutions of Tris 0.2 M and HC1 0.2 M were prepared . F i f t y ml of the forme~ was mixed with 41.4 ml of the la t ter , and the solution was diluted wi th wa te r to a total of 200 ml. A 1:100 dilution of the bu f fe r in wa te r was used in the assay wi th chondroi t inase A,C.

Fris-HCl buffer pH 8. The same stock solutions as indicated above were used. F i f t y ml of the 0.2 M solution of Tr is was added to 26.8 ml of 0.2 M HC1. The solution was then diluted with wa te r up to 200 nil. A 1:100 dilution of the bu f fe r in wa tc r was used in the assay with chondroi t inase A,B,C.

METHOD

"The t issues to be ana lysed were de fa t t ed wi th acetone, acetone-ether , and f inal ly e ther . The t issues were s tored in a s tainless-steel vacuum desiccator (Nikor tanks , Cat. No. 800) to cons tan t weight . The dry defa t t ed t issue weight was regis tered as a re fe rence pa ramete r . For ext rac t ion of AMP, the dry defa t t ed t issues were submit ted to papa in digestion in a medium of distilled wa te r b rought to pH 6.0 with sodium hydroxide and conta in ing 0.005 M cysteine-HCl and 0.005 M disodium versena te ( E D T A ) . Papa in was added in the amount of 50 ?g per mg dry defa t t ed t issue and the t issues were incubated overnight a t 58 ° . If, nex t day, an incomplete solubilization was noticed, papain was added over again and incubation continued fo r 12 addit ional hours. The samples were then dialyzed aga ins t distilled wa te r overnight .

Ten to 30 ~l of the papain digested mater ia l conta in ing 2.5 - - 10 ~g uronic acid or 10 - - 30 ~g pure individual AMP or mixture was added to the column. The column was then eluted wi th 0.02 N NaC1 in 10% CPC; 0.005 N HCI; 0.5 N HCl; 2.0 N HCl; 3.0 N HC1; 4.0 N; 5.0 N and 6.0 N HCl. One ml of each e luent was added in f rac- tions of 0.5 ml. The eluted mater ia l was collected and analyzed fo r uronic acid and neut ra l sugar . Uronic acid was determined according to the m-hydroxydiphenyl method ~4~ on 0.2 ml of the elution collected in every tube. Neu t ra l sugar s were assayed on 0.25 ml of the eluted mater ia l . The an th rone rear-tion, as descr ibed by Drey- wood ~s) was used for the detection of KS, a unique A M P which does not contain uronic acid, but galactose instead. Aliquots of the samples were submit ted direct ly to the f rac t iona t ion procedure or to f rac t iona t ion a f t e r being t rea ted for 15 min with chondroi t inase A,C, a t d i f f e r en t t empera tu res , i.e. 1.5", 4 ° , 22" and 37 ° . The digestion mix ture contained 0.0025-0.005 U of the enz.vme and 20 .~g of A M P (or samples conta in ing between 2.5 and 10 ~g UA) in a f inal volume of 50 ~l. A f t e r the enzymat ic digestion the samples were passed through a celite colunln.

Effec t of test icular hyaluronidase diges t ion on the elution pa t te rn of AMP. The e f fec t of previous tes t icu lar hyaluronidase digest ion was tr ied on pure sample~ of hyaluronidase-sens i t ive and hya lu ron idase - re s i s t an t AMP as well as mix tures of d i f f e ren t pronor t ions of both. The samples were dissolved in distille~ wa te r an~ b rough t to pH 7, the same bein~ valid for the enzyme. Incubat ion w a s per formed at 37" for 30 rain. 1, 2 or 12 hours. The samples were then submit ted to the f rac t ionat ion pro-

cedure. Comparison was made with the procedures of Svejcar and Rober tson ~2~ and Thunell et al "~. Determin- ation of uronic acids was pe r fo rmed according to Bi t te r and Muir cs' when these procedures were assayed. Samples of hyaluronidase-sens i t ive AMPs, submi t t ed to the action of the enzyme for d i f f e r en t l eng ths of t ime, were also f rac t iona ted by our procedure and with the e lut ing solu- t ions used by Svejcar and Rober tson '2' and Thunell et al "~' with the purpose of de te rmin ing the inf luence of the degree of polymer iza t ion on the method.

Ef fec t of undersulphat ion and oversulphat ion of chondroi t in su lphate on the elution pa t te rn . To evaluate the importance of the degree of sulphat ion fo r the complex fo rmat ion with CPC, samples of undersu lpha ted and oversu lphated chondroi t in su lphates were passed th rough the celite column and eluted with the previously mentioned e lu t ing solutions.

Ef fec t of chondroi t inase A,C on the elution pa t t e rn of AMP, pure or in mixture. Twenty ~g AMP was submit ted to the action of 0.025 i 0.0050 U of chondroi t inase A,C per 40 ~l o f water . The incubation was per fo rmed at d i f f e r en t t empera tu re s , i.e. 1.5 ° , 4 ° , 22 ° and 37 °, for d i f f e r en t lengths of t ime in the presence or in the absence of sodium ace ta te a n d / o r Tris buffer~6L The samples were submit ted to the micro-method for f rac t iona t ion of AMP described above. F i f ty ~l was used fo r the f/-ac- ~ionation. The recovery yield and the f rac t iona t ion pat - tern were studied under the ment ioned conditions. The e f fec t of d i f f e r en t pH in the presence or absence of sodium ace ta te a n d / o r Tris buf fe r on the assay was also tried. Complete degrada t ion was considered to have occurred when the mater ia l reac t ing as uronic acid was only p r e s en t in the f i r s t eluent, i.e. 10% CPC in 0.02 N NaC1, which would indicate t h a t the mater ia l could not be precipi ta ted by the qua t e rna ry ammonium compound solution.

Ef fec t of chondroi t inase A,B,C on the elution pa t te rn of AMP. Chondroi t inase A,B,C, 0.020 U per 40,,1 in the presence of 40 ~1 Tris-HCl buf fe r pH 8 and 80 ill H~.O were added to 80 ~g AMP in 40 ~1 w a t e r a t 37 ° fo r 10 min. The elution p a t t e rn was compared with the same mixture holding the same volume of enzyme which was previously inact ivated by hea t ing a t 100 °, and in the absence of the enzyme. F i f ty ~1 o£ the f inal volume of 200~1 was used for the f rac t ionat ion. The sh i f t i ng of the elution to a less concent ra ted HCI e luent was the pa ra - me te r of degradat ion.

Effect of ehondrosulphatases 4 and 6. Pure samples of AMP or mix tures were submit ted to the action of chon- d resu lpha tase 4 or chondrosu lpha tase 6. These enzymes were added to polymerized AMP as well as to mater ia l p r ev ious ly degraded.

Comparison of d i f f e ren t procedures for de te rmina t ion of keratosulphate in a mixture of AMPs. Dete rmina t ion ~f neutra l slzgars '4~ was pe r fo rmed on aliquots of the dif- f e ren t elution subs tances in case samples of pure KS or KS in mix tures with o ther AMPs were submit ted to the f rae t ionat ion procedures of Svejcar and Rober tson '2~, Thunell et al ''~ and the procedure repor ted herein.

Frac t iona t ion of a mixture of HA and CSB previously submit ted to enzymat ic digest ion with test icular hyaluronidase. 5 ~g HA was mixed with 5, 10 and 15 ~g of CSB, and 1.25 l~g tes t icu lar hya luronidase per 5 ~g A M P was added. The subs t ra tc and enzyme were dissolved in distilled w a t e r and were incubated at 37 ° for 30 minutes . Then, they were passed th rough a 1 cm celite column previously equi l ibrated with 10% CPC in 0.02 N NaCI. and eluted as indicated above. Samples of HA and CSB of the same concentra t ion as in'dicated for the mix tu res were run separate ly .

Frac t iona t ion of a mixture of HA, CSA and CSC previously submitted to enzymat ic dit~estion with test icular hyaluronidase. Mixtures of HA, CSA, and CSC were submit ted to enzymat ic di_~estion with tes t icular h.val'lron- idase. The relat ive propor t ions of HA, CSA and CSC were

MICROMETHOD FOR

as indicated above for HA and CSB. The incubation conditions were also similar. Separate samples of HA, CSA and CSC were run simultaneously.

Percentage distribution of AMP's in tissues of 19-wevk- old human embryo. Papain digested material from umbilical cord, articular and epiphyseal cartilage (tibia) and skin, prepared as indicated above, was submitted to fractionation. Samples containing 2 to 5:xg uronic acid per 50 ~l were passed through a 1 cm celite column. The eluents were, as usual, 0.5 ml of the previously mentioned solutions.

RESULTS

Highly reproducible resul ts were obtained for the f rac t iona t ion of AMPs with the micromethod presented.

Keratosulphate was separated from mixtures with other non-sulphated or sulphated AMPs. I t elutes together with the monosulphated anionic macromolecules. The use of the an th rone react ion allows the de te rmina t ion of the galactose moiety of kera tan sulphate. A blank wi thout addi t ion of the reagent , i.e. only H~SO., of the same concent ra t ion as used for the solution of anthrone, was used.

One hundred t~g KS in 50 #l solut ion was passed through a 3 em celite column. Elu t ion was performed with 0.5 ml of the above indicated eluents. Aliquots of 250 /tl were used for the quan t i t a t ive assay of neut ra l sugar with the an th rone reagent. The standard used was 12.5 /~g galactose in 250#1 water . The recovery obtained was 97 ---+ 4%. Aliquots assayed with the m-hydroxydiphenyl reaction did no t show any chromogen, i .e. the m-hydroxydiphenyl reaction is qui te specific for uronic acids. On the other hand, in case aliquots were assayed wi th the carbazole reac- t i o n " a chromogen with a peak of max imum absorp- tion at 530 nm developed, a l though no uronic acids were present in the sample.

Elut ion pattern of AMP

The complex of HA-CPC was split by 0.5 N HCI. Consequently, HA was eluted with tha t solution.

FRACTIONATION 11

Under-sulphated chondroi t in sulphate was eluted with 1, 2 and 3 N HC1, while normal ly sulphated CSA and CSC were eluted with 2 and 3 N HCl. CSB was eluted with 3 and 4 N HCI, oversulphated chondroi t in sulphate with 2, 3 and 4 N HC]. HMS was elut'ed by 2, 3 and 4 N HCI, and Hep with 5 and 6 N HCl. KS f rac t ions were eluted with 10% CPC in 0.02 N NaCl, and 2, 3 and 6 N HCI as shown by the an throne reaction. The recovery of AMP was 100 - 9% (Tables 1 and 2).

E v a l u a t i o n of chondro i t i na se s and chondrosu lphatase 4, 6

The enzyme Ch-ase-A, 0 has shown more subs t ra te specifici ty for HA than for CSC as evidenced by the presence of UA con ta in ing mater ia l in the f i r s t eluent, i.e. 10c~ CPC in 0.02 N NaC1. There is reduc- t ion of the chromogen obtained a f te r the enzymat ic action of Ch-ase A, C, i.e. when complete depolymerizat ion is obtained the chromogcn eluted with 107~ CPC in 0.2 N NaCI is 66+-2G for HA and 44-----1% for CSC as subst ra te , respectively.

Although, according to Miles' in fo rmat ion sheet, it is desirable to keep the solution of Ch-ase A, C, in the re f r igera tor , no effect on its depolymerizing act ivi ty was observed a f te r keeping this enzyme sample deep frozen. A comparison was made of the HA digest ion by two aliquots of the enzyme, one kept at 4 ° , the other defrosted a f te r having been kept frozen at --20 ° for 3 days,. Incuba t ions were performed at 1.5 ° , 4 ° , 22 ° and 37 ° for 15 min. The depolymerizat ions were s imi lar whether deep frozen or at 4 °.

When HA, CSA, CSB or CSC were submi t ted to the influence of Ch-ase A,B,C, under the condit ions indicated by Yamaga ta et al "~*, i.e. 10 minu tes ' incubat ion at 37 ° in the presence of 0.005 U of the enzyme, little effect was observed as indicated by the sh i f t to elution with a lower concentrated eluent. Only in case incubat ion was performed for 16 hours

TABLE 1

F R A C T I O N A T I O N D I A G R A M

Eluents

0.02 N NaCI in 10% CPC

0.005 N HCI 0.5 N HCI 1.0 N HCI 2.0 N HCI

3.0 N HCI

4.0 N HCI 5.0 N HCI 6.0 N HC1

UA

HA UCS

Direct fractionation

CSA, CSC, HMS UCS

CSA, CSB, CSC, HMS UCS CSB, HMS Hep Hep

NS

KS

KS

KS

KS

1 . 5 °

UA

HA (CSA. CSC)

Fracfionation after digestion with Ch-ase A, C (15 rain)

I 4 °

UA

~ HA (CSA, CSC)

CSA, CSC, HMS

CSA, CSB, CSC, HMS

CSB, HMS Hep Hep

KS CSA. CSC, HMS

KS CSA, CSB, CSC, HMS CSB, HMS Hep

KS Hep

22 ° - - 37 °

NS UA

KS I HA, CSA, CSC I (CSB)

KS HMS

KS CSB, HM.S

CSB. HMS Hep

KS Hep

m

KS

KS

KS

KS

UA - - Uronic acid NS = Neutral sugar UCS = Undersulphated chondroitin sulphate Parenthesis indicates trace amounts

12 BLUMENKRANTZ and ASBOE-HANSEN

TABLE 2

ELUTION PATTERN OF DIFFERENT AMPs. PERCENTAGE RECOVERIES AS UA (UA-CONTAINING AMP) OR GALACTOSE (KS) ARE INDICATED. EACH VALUE REPRESENTS AN AVERAGE OF SIX DETERMINATIONS. THE ADDED AMOUNT WAS CONSIDERED 100~7o.

Eluents

0.02 N NaC1 in 10% CPC 0.005 N HC1 0.5 N HC1 !.0 N HC1 2,0 N HC1 3.0 N HC1 4.0 N HC1 5.0 N HC1 6.0 N HC1

HA

97 ::1:4

59 ± 2 4 1 4 - 2

B

1 8 ± 2 8 0 ± 3

CS

C

4 6 ± I 53 ~ 2

Under- sulph.

59 ± 2 21 -4-2 1 9 + 1

Over- sulph.

4 ± 1 59 ~-2 37 ± 2

HMS

52 ± 2 3 2 ± 1 14 :i: 1

Hep

28 ± 2 60 :~2

KS

28 ± 2

35 ± 2 21 -4-2

1 5 + 2

at 37 ° a consistent depolymerization of the order of 90% for CSC and 20% for HA was observed. The need of Tris buffer pH 8.0 and sodium acetate for the enzymic action to proceed was confirmed insofar ~s no depolymerization was observed in water or in buffer alone. The enzyme was not active in solution beyond one week. The recovery, as calculated by the ~ronic acid content of AMP samples incubated overnight with or without Ch-ase A,B,C, was 100 ---- 2%.

In our system, chondrosulphatases 4 and 6 were ~hown to be non-active on the polymers CSA, CSB ~nd CSC. When the samples were previously submitted to the action of Ch-ase A, C, for 10 rain at 37 ° and afterwarrds to the chondrosulphatases 4 or 6, the ohly compound remaining almost fully polymerized was CSB.

F~ffect of Tris buf fer and sodium acetate ~ n the act iv i ty of Ch-ase A, C

No difference in the percentage of degraded HA, .CSA and CSC was observed whether the digestion mixture contained sodium acetate and/or Tris buffer o r the enzyme was acting in plain aqueous medium ,(Table 3). Comparisons were made in relat ion to temperature, i .e. 1.5 ° , 4 ° , 22 ° and 37 °, and to t ime of incubation in the presence or absence of buffer and :sodium acetate. The degradat ion increased with in- .creasing temperature or t ime of incubation (Table 4). No differences were noticed when samples were ~incubated at the same temperature or for the same length of t ime in the presence or absence of ei ther T r i s buffer or sodium acetate or both.

The effect of decreasing amounts of Ch-ase A,C, on the depolymerisation of HA and CSC is shown in 'Table 5.

E f f ec t of AMP res i s tant to Ch-ase A, C ,on the degradat ion of CSA and CSC

When mixtures of CSA or CSC with HMS or KS were submitted to degradat ion with Ch-ase A, C, in -the presence or absence of Tris buffer and sodium ,acetate c*~ no inhibit ion of the enzymatic degradat ion o f e i ther CSA or CSC was observed.

Effec t of pH on the e n z y m i c act iv i ty of Ch-ase A, C

No difference in the sensi t ivi ty of HA, undersulphated chondroitin sulphate and CSC was observed when assaying the effect of pH from 6.0 to 8.0

Ef fec t of a hya lu ron idase - res i s t an t AMP on the degradat ion of HA

Samples of HA and CSB run separately or as mixtures without enzyme were eluted with 0.5 N and 3-4 N HCI, respectively. The recoveries obtained were 90 ----- 5%.

The presence of a hyaluronidase-res is tant AMP (CSB) inhibited the degradat ion of HA by the enzyme. The elution pat tern showed that, a f te r incubation with the enzyme for 30 min, HA was eluted with 0.5 N HCI while the CSB of the mixture was eluted with 3 and 4 N HCI. When mixtures of HA and CSC were influenced by hyaluronidase and then fract ionated, most of the HA and CSC turned out to be degraded, as shown by uronic acid containing mater ial being eluted with all the eluents from 10% CPC in 0.02 N NaC1 to 3 N HC1, the uronic acid mater ial eluted with the la t te r being minimum (Table 6). The recoveries obtained were 92 ± 4%. Undepolymerized CSC was eluted with 2-3 N HCl only.

Ana lys i s of AMPs from h u m a n embryon ic t i ssues

A predominance of HA was found in umbilical cord and skin, while chondroitin sulphates predominated in car t i lage (Table 7).

DISCUSSION

The method presented offers the following ad- vantages to the most widely used procedures for fract ionat ion of AMPs. 1) The precipi tat ion and fract ionat ion is performed on a s tandard disposable column; 2) the results are highly reproducible; 3) HCl is a most convenient eluent, especially because it allows fur ther hydrolysis in case determinat ion of hexosamines is also desired. The concentration of

M I C R O M E T H O D F O R F R A C T I O N A T I O N 13

TABLE 3

EFFECT OF INCUBATION WITH Ch-ase A, C AT 1.5 ° FOR 15 MIN ON THE CONVERSION OF HA, CSA, CSC, UNDERSULPHATED CS AND OVER- SULPHATED CS (UCS AND OCS) TO UNSATURATED DISACCHARIDE, WITH AND WITHOUT ADDITION OF SODIUM ACETATE AND TRIS BUFFER. THE VALUES REPRESENT PERCENTAGE DISTRIBUTION OF UA.

Eluents

0.02 N NaC1 in 10% CPC 0.005 N HC1 0.5 N HC1 1.0 N HC1 2.0 N HC1 3.0 N HC1 4.0 N HC1 5.0 N HC1 6.0 N HC1

Without Na-acetate

or Tris

HA

59 4 - 2

2 5 - + I

With Na-acetate and /or Tris

HA

59 - + 2

25 4 - 2

CSA

2 4 - 0 . 1

CSC

11 4 - 0 . 5

UCS

31 ± 2 44-+1 1 0 - 4 - 1 1 5 - + 1

4 3 4 - 3 53 - + 3

4 9 - + 1 4 0 4 - 1

OCS

9 - + 2

45 4 - 2 50 + 3

TABLE 4

EFFECT OF IN(~UBATION OF AMPs WITH Ch-ase A, C FOR 60 MIN AT DIFFERENT TEMPERATURES.

Eluents

0.02 N NaC1 in 10% CPC

0.005 N HCI 0.5 N HCI 1.0 N HCI 2.0 N HCI 3.0 N HCI 4.0 N HCI 5.0 N HCI 6.0 N HCI

1.5 ° HA CSC CSB

5 6 - + 2

6 -+6.2

38 4- I

2 4- 0.02 1 8 - + 1 6 4- 0.5 20 ± 1

8 0 - + 1

4 ° HA CSC CSB

6 7 - + 2 4 2 - + 3 8 - + 1

1 2 4 - 2 2 4- 0.1 12 -+ 1

8 0 4 - 4

22 ° HA CSC CSB

67 ± 3 42 4- 2 12 4- 2

1 0 - + 1 78 - + 2

37 ° HA CSC CSB

68 -+ 3 42 4- 2 14 ± 3

1 0 + 2 78 - + 4

TABLE 5

EFFECT OF DECREASING AMOUNTS OF Ch-ase A, C ON THE DEPOLYMERiZATION OF HYALURONIC ACID AND CHONDROITIN SULPHATE C. THE INCUBATIONS WERE PERFORMED AT 15 ° FOR 15 MINUTES. INITIAL AMOUNT OF THE ENZYME WAS 0.005 U. DILUTIONS WERE MADE WITH DISTILLED WATER. VALUES REPRESENT PERCENTAGE UA FROM HA, AND, WITHIN BRACKETS, UA FROM CSC.

Eluents

0.02 N NaC1 in 10% CPC

0.005 N HCI 0.5 N HC1 1.0 N HC1 2.0 N HC1 3.0 N HC1 4.0 N HC1 5.0 N HC1 6.0 N HC1

Initial 0.005 U

70 - + 3 (25 -+ 2)

30 4 - 2

(36 4- 1) [39 -+ 2)

1 + 1

5 2 4 - 2

4 8 - + 3

(46 -+ 2) (54 4- 3)

I

With Ch-ase, A, C Dilutions

1 + 5 1 + 1 0

4 1 4 - 1 3 0 - 4 - 3

5 9 4 - 2 7 0 4 - 3

(30 4- 1) (27 4- 2) (70 -+ 2) (73 -+ 2)

1 + 5 0

2 4 - 0 . 1

98 4 - 3

(23 -+ l) (77 -+ 2)

1 + 1 0 0

1 4- 0.05

99 4 - 3

(26 4- 1) (74 -4- 1)

1 + 2 5 0

0

100 4 - 4

(28 -4- 2) (72 -+ 2)

Without Chase , A C

0

100 4 - 4

(29 + 1) (7l ± 1)

TABLE 6

EFFECT OF THE ACTION OF TESTICULAR HYALURONIDASE ON THE ELUTION PATTERN OF AMPs. 1.25 ~g OF ENZYME PER 5.0 ~g AMP, BOTH DISSOLVED IN WATER, WERE INCUBATED AT 37 ° FOR 2 HOURS. THE FIGURES REPRESENT PERCENTAGE OF DISTRIBUTION OF UA.

Eluents HA CSC CSB HA + CSC + CSB HA + CSB CSC + CSB

0.02 N NaC1 in 10% CPC 0.005 N HC1 0.5 N HC1 1.0 N HC1 2.0 N HC1 3.0 N HC1 4.0 N HC1 5.0 N HC1 6.0 N HC1

1004-t-5 1 5 4 - 1 5 4 - 0 . 2 7 4 - 0 . 1

37 - + 2 2 6 4 - 1

2 -+ 0.02

42 4 - 2 5 6 4 - 1

2 2 - + 1 4 ± 0.02

1 7 - { - 1 9 -+0 .2 9 ± 0 . 1

13 ± 0.5 26 4- 0.2

32 - + 2

24 - + 2 4 3 - + 1

2 2 4 - 1

1 5 ± I 2 6 ± I 1 5 + 2 2 2 ± 1

HA + KS

45 4 -2 .

[ 55-4- I

14 BLUMENKRANTZ and ASBOE-HANSEN

TABLE 7

FRAL-"I'IONATION OF BIOLOGICAL MATERIAL. TISSUES FROM HUMAN EMBRYOS SUBMITTED TO AMP FRACTIONATION. RESULTS REPRESENT PERCENTAGE DISTRIBUTION OF URONIC ACID CON- TAINING MATERIAL ELUTED WITH VARIOUS SOLVENTS.

Eluents

10~ CPC in 0.02 N NaC1

0.005 N HC1 0.5 N HC1 1.0 N HC1 2.0 N HC1 3.0 N HC1 4.0 N HC1 5.0 N HC1 6.0 N HC1

Percentage distribution of UA

Umbilical cord

71

7 22

Cartilage ,

Ski__~n Articular Epiph)scal

54 I 12 / 11

3~ 67 71 21 18

HC1 in the eluent~ may be changed to one which is normal ly des i rable for hydrolys is , and the samples may be hydrolyzed wi thou t previous t r e a tmen t . In o ther procedures in common use for f r ac t i ona t i on of n l ic ro-quant i t ies of AMPs , as e.g. tha method of Thunell et al '~'', a s tep of alcoholic p r ec ip i t a t i on of the sodium and magnes ium sal ts of A M P s eluted f rom the cellulose columns mus t be pe r fo rmed before hydrolysis. I t is obvious, however, that , work ing wi th small quan t i t i e s of AMPs , the fewer s teps the procedure includes, the be t t e r recover ies m a y be expected. Thunell et al TM indica te tha t , if any t u rb id - i ty appea r s d u r i n g the elut ion of the A M P s f rom the cellulose column, i t may be e l imina ted wi th 4 M MgCl~ or a s a t u r a t e d solut ion of NaCI, and the con- t en t of the tubes belonging to the same e luent m a y be pooled. Accord ing to our exper ience, a possible t u rb id - i ty is due to incomplete adsorp t ion and re ten t ion of the complex onto the column. A t u r b i d i t y may rep- r esen t pa r t i a l or to ta l loss of a f r ac t ion f rom the cor responden t posi t ion. In case t u r b i d i t y occurs, the samples should be r e -ch romatographed .

The use of the m-hydroxyd ipheny l assay fo r de te r - mina t ion of uronic acid is handy because of the small amount of sample t ha t is requi red and the quickness of the procedure . Besides, th is method has a h igh spec i f ic i ty , and KS does not i n t e r f e r e wi th the detec- t ion of U A - c o n t a i n i n g AMPs . The poss ib i l i ty of de tec t ing KS toge the r wi th the o ther A M P s renders th is f r ac t iona t ion procedure useful for s tudies of va r ious normal or pathological processes where CS a re subs t i tu t ed by KS. When KS was passed th rough the column and eluted a s indica ted by Sve jca r and Rober tson "~' and Thunell et al '~' a very low recovery of KS was obta ined due to the development of a yellow colour wi th anthrone, which probab ly masked the chromogen of neu t r a l sugars .

The use of d i f f e r e n t concent ra t ions of HCl to s epa ra t e non-sulphated and su lphated A M P s f rom the i r complex wi th CPC has the advan t age of need ing fewer solut ions to be p r e p a r e d than o ther p rocedures and even y i e ld ing h igh ly reproducib le resul ts . The procedure repor ted by Thunel l et al '~ uses 1) 1%

CPC; 2) 0.3 M NaCI 3) 0.28 M MgCI:; 4) n -propanol - methanol -ace t ic ac id ; 5) 0.75 M MgCh in ace t ic ac id ; 61) 0.9 M MgCl... in acet ic acid, and 7) 0.75 M neu t ra l solut ion of MgCl~. Sve j ca r and Rober t son '-~* use the same e lu t ing solut ions except solut ion 3 which is 0.30 M MgCl.., ins tead of the 0.28 M of Thunel l et al ''~'. Solut ion 6 is 0.75 M neu t r a l MgCl~ while solu- t ion 7 is 1.25 M neu t ra l MgCl2. Accord ing to S v e j c a r and Rober t son '2' the A M P s eluted are H A wi th solu- t ion 1 and 2, CSA wi th solut ion 4, CSC wi th solu- t ions 4, 5 and 6, CSB wi th e luents 5 and 6 and HMS wi th e luent 3. Acco rd ing to Thunel l et al ̀ '~ H A is eluted wi th solut ion 2; CSA and CSC wi th so lu t ions 4 and 5, while solut ion 6 elutes CSB. These au tho r s do not d i s r e g a r d con tamina t ion wi th the o the r chon- d ro i t in su lphates . Solut ion 7 can elute a m i x t u r e of ga l ac tosaminog lycans while solut ion 3 elutes HMS. The method of Thunel l et al ''~' and tha t of Sve jca r and Rober tson '2' use p rac t ica l ly the same ser ies of eluents , and they requ i re a more labor ious p rocedure than t ha t p resen ted in th is paper . The elut ion of an indiv idual AMP wi th more than one e luent m a y express po lyd i spe r s i ty a n d / o r d i f f e r e n t degrees of su lphat ion . This is shown by our expe r imen t s of t ime-course d e g r a d a t i o n wi th t e s t i cu l a r hya lu ron i - dase and f r ac t i ona t i on of a pu r i f i ed undersu lpha ted chondro i t in sulphate . As shown by Meyer et a l '~', chondro i t in su lpha tes A and C f rom d i f f e r e n t sources possess a cons iderab le degree of po lyd i spe r s i ty and, a t leas t in some t i ssues , a v a r y i n g degree of su l - phat ion.

Compar ison of the elut ion p a t t e r n s of hya lu r - on idase-sens i t ive A M P s d iges ted d u r i n g d i f f e r e n t lengths of t ime shows tha t these A M P s a re e luted different ly• in re la t ion to the d e g r a d a t i o n process. This observa t ion is val id when us ing the e luents of Thunell et al '"' and Sve jca r and Robertson"- ' as well as ours (Table 1~. I t shows t ha t i t is p rac t i ca l ly im- possible to assess the type of A M P eluted only by the e luent and the d e t e r m i n a t i o n of hexuron ic ac ids TM.

The same is t rue of the o the r way used to d e t e r m i n e the type of A M P by a ce r t a in e luent and the presence of g lucosamine /ga l ac to samine ~>, p a r t i c u l a r l y i f one cons iders the over l app ing of some A M P s eluted by more than one of the e luents '~' '~

The rron-homogeneous p a t t e r n of A M P elut ion a f t e r t e s t i cu la r hya lu ron idase d iges t ion m a y be explained by the mechanism of act ion of testict~lar hya luron idase , be ing an enzyme wi th both hydro - lyt ic and t r ansg lycos idase Pction,9. ,o, The main product of t e s t i cu l a r hya lu ron idase d iges t ion of HA, CSA and CSC is a t e t r a saccha r ide , a l though a ser ies of o l igosacchar ides , r a n g i n g f rom di- to octo-sacchar ides , have in fac t been isolated '" ' ,o,. The above ment ioned t r ansg lycos ida t i on occurs when both the h ighe r po lymers and the o l igosacchar ides a re submi t t ed to the act ion of hyaluronidase ' : ' - ,o , The lack of r e ten t ion of the UA con ta in ing m a t e r i a l by the column indica tes the depolymer iza t ion . As the end product of Ch-ase A, C, deg rada t i on is an u n s a t u r a t e d d i sacchar ide '~', i t may be concluded t h a t the dis- acchar ide cannot fo rm an insoluble complex wi th the q u a t e r n a r y ammonium compound.

MICROMETHOD FOR FRACTIONATI,ON 15

Our resu l t s a re in accordance wi th those of L i n k e r et al '1' ' in tha t , a f t e r complete deg rada t i on (all ma- t e r i a l e luted wi th 0.02 N NaCl in 10% CPC) , a r educ t ion in the UA chromogen in the o rde r of 66 ----- 2% for H A and 44 ---- 1% for CS was found. The f ac t t h a t a small p ropor t ion of CSB is depolymer ized by Ch-ase A, C may be expla ined by the presence of a hyb r id region con t a in ing g lucuronic ins tead of iduron ic acid. Cons ide r ing the i n h i b i t o r y e f fec t of CSB on the d iges t ion of HA by t e s t i cu l a r hya lu ron idase i t seems wor thwhi l e no t i c ing t h a t the use of th i s p a r t i c u l a r enzyme in s e p a r a t i n g m i x t u r e s ~)f hya lu ron idase - sens i t i ve f rom r e s i s t a n t A M P s may involve se r ious p i t fa l l s .

ACKNOWLEDGMENT

The authors acknowledge the assistance of Mrs. Hanne Boyden.

exper t technical

REFERENCES

1. Schiller, S., Slover, G. A., and Dorfman, A. (1963). J. Biol. Chem. 236, 983-987.

2. Svejcar, J., and Robertson, W. van B. (1967). A~ml. Biochem. 18, 333-350.

3. Thunell, S., Antonopoulos, C. A., and Gardell, S. (1967). J. Atheroscler. Res. 7, 283.

4. Blumenkrantz, N. and Asboe-Hansen, G. (1973). Anal. Biochem. 54, 484-489.

5. Dreywood, R., (1946). Ind. lng. Chen~. (anal. Ed.) 18, 499.

6. Yamagata, T., Saito, H., Habuchi, 0., and Suzuki, S. (1968). J. Biol. Chem. 243, 1523-1535.

7. Bitter, T. and Muir, H. M. (1962). A~zal. Biochem. 4, 330-334.

8. Meyer, K., Davidson, E., Linker, A., and Hoffman, P. (1956). Biochim. Biophys. Acta 21, 506-518.

9. Weissman, B. (1955). J. Biol. Chem. 216, 783-794. 10. Hoffman, P., Meyer, K. and Linker, A. (1956). B/o-

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Micromethod for fractionation of acid mucopolysaccharides