Acrylamide gel electrophoresis of acid glycosaminoglycans for the study of molecular size distribution

ANALYTICAL

52, 382-394

BIOCHEMISTRY

Acrylamide

Gel

(1973)

Electrophoresis

Glycosaminoglycans of D. HSU, Resenrch

Zmtitute

Molecular

for Size

of the

Study

Distribution1

P. HOFFMAN,

AND

T. A. MASHBURN,

of the Hospital of Medicine, New

for

Joint Diseases, Mount New Pork 10035

Received

August

York,

11, 1972;

Acid

accepted

October

JR.’ Sinai

School

17,. 1972

Chondroitin sulfates from bovine nasal septum and lamprey cartilage and keratan sulfate from human costal cartilage show a wide molecular size polydispersity by acrylamide gel electrophoresis. The number of fractions were limited only by the number of slices obtained from gel slabs nftel elertrophoresis. The polysaccharides are probably distributed in a continuum of varying chain lengths. For the study of the size distribution of sulfated glucosaminoglycnn by acrylamide gel disc electrophoresis, several buffer systems were formulated according to a general guide.

The charge density of an acid glycosaminoglycan is determined primarily by the charge:mass ratio of the repeating disaccharide unit, and independent of chain length. Consequently, electrophoretic mobility only reflects molecular size variations if molecular sieving effects arc operative. Thus, glycosaminoglycans, which on conventional electrophoresis run as single bands (1), can he spread extensively on polyacrylamide gel electrophoresis. The sieving capacity of polyacrylamide gel has been utilized for molecular weight determinations of acid glycosaminoglycans (2,3). It was shown that sulfated glycosaminoglycans each gave a single but rather wide band. The band width was attributed to polpdispereity (2). In this paper, we describe the subfractionation of the wide band into many reproducibly contiguous bands indicating that the polydispersity is due to variations in chain length. The higher resolving capacity of gel disc electrophoresie compared to conventional gel electrophoresis results from the use of a discontinuous buffer system which provide, q a moving electrolyte boundary between leading and trailing ions. A sample having an elrctrophoretic ‘This investigation was Public Health Service. ’ Established Investigator Copyright All rights

supported of the

by

Grant

American Heart 382 @ 1973 by Academic Prrss, Inc. of reproduction in any form reserved.

AM08482

from

Association.

the

Unitctl

States

ELECTROPHORESIS

OF

GLYCOSAMINOGLTCANS

383

mobility between the leading and the trailing ions is sandwiched by the boundary creating an extremely sharp starting zone (4,5). Hjertkn et al. (6) sharpened the starting zone by applying samples in a solution which had a lower conductivity than the buffer, and stated t,hat although the two techniques have the same resolving power, a continuous buffer system might be more advantageous than a discontinuous buffer system because t#he possibility of varying the pH and the composition of the continuous buffer was more flexible. However, as far as the capacity for sharpening the starting zone is concerned, the discontinuous buffer system is more effective, especially for preparative runs, where the sample must be applied in a larger volume. Therefore, several discontinuous buffer systems were devised according to the principles of Ornstein (7) and applied to the electrophoresis of sulfated glycosaminoglycans. In the systems generally used for the gel disc electxophoresis of proteins, sulfated glycosaminoglycans run as a concentrated, single band sandwiched between the leading and the trailing anions, and do not spread t.o the extent noted in the systems described in this paper. MATERIALS

AND

METHODS

The sulfated glycosaminoglycans used have been previously described (1). Ch4-S3 was obtained from bovine nasal septum after papain digestion or alkali treatment followed by purification by free-flow electrophoresis (8). The purified fractions have been different,iated by the terms papain-t’reated and alkali treated Ch4-S. Lamprey CM-S was obtained by treatment of lamprey cart.ilage with pepsin, trypsin, and panprotease as preriously described (9). KS was a purified fraction from human costal cartilage (10). The ChG-S sample was obtained from shark scapular cartilage by alkali treatment and purified by free-flow electrophoresis. The amino acid levels were determined by Beckman Amino Acid -\nalyzer (11). Papain-treated Ch4-S contains 2.8% amino acid; alkali treated Ch4-S, 0.75% amino acid. Preparation of Gels. Separating gel was prepared by mixing 1 volume of water and the following stock solutions: 1. 1 volume of buffer 4 times the required final concentrations. 2. 1 volume of 30% (w,/v) acrylamide and 1% (w/v) methylene bisncrylamide in water. 3. il half volume of TEMEII (0.06 ml in 12.5 ml water). 4. d half volume of ammonium persulfate (70 mg in 12.5 ml water, prcparcd fresh each week). a Thr nhbrel-intions used : Ch4-S. t~is(h~drospmethyl)aminomethane; mine: RPR. hromphenol blue.

chondroitin TEMED,

4-sulfatr : KS, krratan sulfate W.N.N’,N’-tptmmethvlethylenedia-

; Tris,

384

HSU,

HOFFMAN,

AND

MASHBURN

For spacer gel, the monomer mixture of 10% acryamide and 2.5% methylene bisacrylamide in water was used. The gel was cast in tubes, 7.5 X 0.5 cm, for analytical runs (Buchler Analyt,ical TemperatureRegulated Polyacrylamide Gel Electrophoresis Apparatus), and in cells (8 cm X 10 cm X 3 mm) for preparative runs (Ortec Slab Polyacrylamide Gel Electrophoresis Apparatus). The separating gel was filled to the height of 5.5 cm in the tubes; and 7 cm in the cells. Spacer gel of about l-cm height was used in both cases. Only the separating gel was used for a continuous buffer system. Buffers for Disc Electrophoresis. Three buffer systems were used. The compositions are listed in Table 1. Chloride ion was selected as the leading anion. The concentration was maintained at 0.05 M for both separating and spacer gels. The concentrations of weak acid and base for the reservoir were calculated according to Ornstein (7). Application of Samples. For analytical runs, the sample was generally applied using discs (4 mm diameter) cut from cellulose polyacetate strips (Sepraphore III, Gelman Instrument Co.). About 2 ~1 of sample in water was applied from a disposable 10 ~1 pipet (Corning) by touching the tip to the disc, and letting it wet by capillary action. It was then placed on the gel which had been blotted dry with tissue paper. Sample was also applied according to the method of HjertGn et nl. (6) to achieve sharpening of the starting zone by a difference in the conductivity of the sample and the separating gel. For disc electrophoresis a sample in a volume of up to 0.1 ml was applied in the spacer gel buffer containing 10% sucrose. For preparative runs in Tris-HCl, pH 8, in order to stack sample into a thin starting zone, the sample was dissolved in 0.5 ml of Tris-borate (0.03 M in boric acid), pH 8, containing 6 M urea, and was introduced

Compositions

TABLE for Senarating

of Buffers0

Separat,ing gel (0.05 Af HCI) Tris

System Tris-HCl-barbital Tris-HCl-glyrine Tris-HCl-boric

acid

(M)

0.086 0.5 0.5

pH 8 9.2 9.2

1 Gel, Spacer

Spacer

Gel,

and Reservoir Reservoi@

gel

10.05 M HCl) Tris

(fir)

0.054 0.07 0.07

12The buffers were prepared at, fourfold concentration * The lower reservoir buffer was sometimes replaced for convenience. This did not, change the electrophoretic

pH 7 8 8

Tris

(M)

0.003 0.026 0.008

Weak aqid i,Ifj

pH

0.033 0.039 0.035

7 8.9 s

for the preparation of gels. with 0.03 Af Tris-HCI, pH 8.2, patt,ern.s.

ELECTROPHORESIS

OF

GLTCOSA31INOGLTCANS

385

through small tubing on to the gel, with the electrolyte already in position. On top of the sample, 2 ml of Tris-borate buffer containing 3 M urea was similarly layered. Urea, instead of sucrose, was used to incrcasc the density. For preparative disc electrophoresis, the sample in the spacer gel buffer containing 20% sucrose was similarly layered on the top of the spacer gel. In every run, a small amount of BPB was added as an indication of the sharpness of initial starting band and as a guide for terminating the run. Electrophoresis und Staining. For analytical runs, a current of 1.5 mA/tube was applied for 5-15 min until the BPB zone had entered the separating gel, then increased to 5 mA/tube for the rest of the run. When the BPB zone reached about 2 cm from the end of the t’ube, the run was terminated. The total time for a run was 2-3 hr for buffers composed of strong electrolytes; about 30 min for weak electrolytes; and 45-60 min for disc clectrophoresis. Gels were stained in 0.1% toluidinc blue 0 for at least 30 min, and destained in running water overnight. For rapid observation of the bands, gels were stained for 5 min only. For preparative runs, in 0.05 1\1 Tris-HCl, pH 8, the clcctrophoresis was carried out at 50V (70 mA/cell) for 5 min than at 1OOV (200 mA/cell) for 2 hr; in the discontinuous buffer system. at 150V (25 mA for 2 cells) for 15 min then at 300 V (50 mA for 2 ~11s) for 50 min. After elect,rophoresis, the gel was sliced wit,11 steel blades spaced about 2 mm apart. The gel slices were crushed by forcing them through a 5-ml disposable syringe and extracted wit,h 0.1 M C&l,. The extracts werr filtered through glass wool, dialyzed, evaporated almost to dryness under reduced pressure at 40°C and diluted to an appropriate volume with water for analytical gel electrophoresis. Alkali Treatment. Ch4-S recovered from preparative gel elect.rophoresis was treated with 0.5 II1 NaOH with or without 0.01 M NaBH, at room temperature for at least 24 hr. The solutions were ncmralized with 1 M acetic acid, and applied using cellulose acetate discs for analyt’ical gel electrophoresis. RESULTS

Continuous and Iliscontinz~ous Buffer Systenls. Alkali treated Ch4-S moved on cellulose polyacctate strip elect,rophoresis as a single discrete band. On polyacrylamide gel with a single buffer, t,he band was very broad and diffuse. This broad band was separated into at least eight contiguous discs by slicing, and each disc was placed on top of a fresh gel column. The contents of each disc migrated discretely and main-

386

HSU,

HOFFMAN,

AND

MAHHBURN

tained the original differences in mobility. The fact that the differences in mobility were maintained reproducibly discounted overloading or methodological artifacts as the cause of band broadening, and indicated real molecular size differences. The fact that a single sharp band was always observed in cellulose acetate electrophoresis discounted charge differences as the cause of band broadening. Since slices from the broad band maintained reproducibly different mobilities in the same system, the isolated slices were used to compare the resolving power of a variety of systems as described below. Alkali-treated Ch4-S (4 mg) was run preparatively in 0.1 M TrisHCI, pH 8. Eighteen 2-mm wide strips of gels were then sliced 2 mm apart from the gel slab starting from the BPB dye front. Alternate gel strips were combined and extracted according to the procedure described in Methods. This sample was diluted to 0.1 ml with water and 2 ~1 was used for each analytical gel electrophoresis. In the continuous buffer systems using either 0.05 M and 0.1 M Tris-HCl, pH 8; 0.1 M sodium phosphate, pH 7.5; 0.05 M Tris-barbital, pH 8; or 0.05 M Tris-glycine, pH 9.6; the sample was separated into 8 bands. The separated bands in the first two buffers were more compact than in the others. The experimental results showed that the buffers composed of strong electrolytes with higher ionic strength gave more compact bands. However, in these higher conductivity buffers electrophoresis must be run at lower voltage gradients, and consequently, for much longer running times. On the other hand, the buffers composed of weak acids and bases gave less compact bands. The technique of HjerGn et al. (6) for concentrating a dilute sample into a thin starting zone gave poor separation in these systems. Therefore, disc electrophoresis was tested. When electrophoresis of this sample was carried out in a discontinuous buffer system according to the technique originally described by Ornstein and Davis (5) for the analysis of protein, six of the eight resolved bands were concentrated in a single zone and migrated wit’h the anion front. The other two resolved bands were smeared above the zone. This indicated t,hat the molecular size differences were small and that a correct choice of buffers was necessary for the sieving effects of the gel to adequately resolve the sample. Increasing the gel concentration to 15% had only a minimal effect on’the resolution. However, elcctrophoresis in either of the three discontinuous buffer systems described (Table 1) clearly maintained the separation of the eight bands. The necessity of a spacer gel for the disc electrophoresis was tested bv running the clectrophoresis in the gels with or without spacer gel. When the above sample was applied in a volume of 0.1 ml in both cases, the tllbe with spacer gel gave better separation (see Fig. 1). However,

ELECTROPHORESIS

OF

GL~COSAMI~O(;LTCA~S

387

FIG. 1. Disc-gel electrophoresis of alkali treated Ch4-S with and without spacer gel. The sample was a combined cstrart, of eight alternate gel slices (2 mm wide) from a slab-gel elcctrophoresis of the C114-S using a continuous buffer system. 0.1 hl TrkHCl, pH 8. The disc-gel rlcctrophoresis was run in the Tris-HCI-glycine system. The sample was applied: A. about 28 pg in 0.1 ml on the spacer gel; B, about 28 pg in 0.1 ml without spacer gel; C, about 14 fig on a ccllulosc polyacetate disc.

a spacer gel is unnecessary when the application volume is sufficiently small. Molecular Size Distribution of Sulfated Glycosaminoglycans. Papaintreated Chili-S and KS (2 mg of each) were run preparatively using the Tris-HCI-glycine system. The gel slabs were sliced 2 mm apart, and the odd and even-numbered slices were combined. The two sets of combined gels were extracted according to the procedure described in Methods. As shown in Fig. 2, the separated bands corresponded to the number of combined slices. This suggested that the broad band resulted from a continuous variation in the number of disaccharide units incorporated into the single chains within the limits of the chain size. which can only bc proved after the isolation of adequate The assumption, amounts of purified fractions, is that chains differing by one disaccharide unit overlap but that more t,han one unit difference is sufficient for complete separation.

388

HSU,

HOFFMAN,

AND

MASHBURN

FIG. 2. Molccul:w size polytlisprrsity of papain-treated Ch4-S and KS. TLC disc electrophoresis was run in the Tris-HCl-glycinc s~ztc>rn. Tllo s:~m~~lcs in a volume of 10 pl wcrc applied without apawr grl. The fscls ~1, B. and C contain (314-S; thr others, KS. The gels A :mtl D contain 50 fig ~a(*11of CIA-9 and KS, rqwctivcly. Thr gels shown with hand scyarnlions are the samplt~s of the c,omhined extracts of odd (B, E) and cvcn (C, F) numbered grl slices. They were sliced from the gtsl slnh after electrophorcsis was cnrriwl out in the Tris-HCl-alycinc system. Thr wide hantls of the gels stnincd blur with toluidinc blurb 0 XP artifact. at, the bottom

When the Ch4-S fractions having slower mobilities were sliced from the gel, extracted, and treated with NaOH as described in Methods, no obvious changes in the elcctrophoretic mobilities were observed, demonstrating that the serinc containing peptide to which the xylose at the linking end of the chain was attached did not, influence the mobility in the system. Other sulfated glycosaminoglycans also showed size polydispersity. As shown in Fig. 3, they also differ in their clectrophoretic mobilities. The thick bands at the bottom indicated that some small molecules with higher mobilities than the effective mobility of glycinate or borate at tl1e running pH of 9.6 were not resolved. The extreme case was the lamprey Ch4-8 which was almost concentrated into a single thick band. This confirms the visaosimetric studies of Mathews (12) that Ch-8 chains from lamprey cartilage proteoglycans are significantly smaller than those from bovine nasal septum proteoglycans. Moclijicatio?l of Buffer Systems for Disc Electrophoresis. To complete resolution of the thick, concentrated zone of Ch4-8 from nasal septum (Fig. 2) requirctl raising the pH in the separating gels. This was done

DS

HS H 6 C ~.“-@----~, -.

CL

FIG. 3. Molecrtl:~r size pol~dispersi1.y of olllei ~uiratctl gl~c~o~;uninogl~~~lll~. The tlix elcctrophoresis WIS rim in the Tris-HCl-boric acid system. Samples (about 50 pg each) were applied in a volume of 10 pl without spnwr gel. The type of glycosaminoglycan is indicated on ihr figure: DS. dermnlan sulfate; NH. heparitin sulfate; H, heparin; C, chondroilin 6-PllIfate; CT,. lnml)rry Cl14S. This buffer system gate the Same results us the Tris-HCI-glycine Pyrtrm. Xa nrtifwtuxl hnnds occur in this system.

by adding more Tris to the buffers or replacing Tris with a stronger base. In the Tris-HCl-barbital system, the band was completely resolved at pH 8.2, while in the Tris-HCLglycine system, the amount, of Tris had to be increased to 1 M. However, under these conditions, part of the lamprey Ch4-S still remained unresolved. To complete this resolution, the pH of the separating gel was raised to 8.7 (0.05 ill HCl-0.3 M Tris) in the Tris-HCl-barbital system. In the Tris-HCl-gIycine system, ethanolamine was used to replace Tris as a base so as to raise the pH to 9.8. As an example, CM-S from nasal septum and lamprey cartilage were fractionated by preparative electrophoresis using the Tris-HCl-barbital system at pH 8.7. Spacer gel buffer was 0.05:11 Tris-HCl, pH 7. The upper reservoir buffer was 0.03 M barbital and 0.06 M Triu, and the lower

390

HSC.

HOFFMAN,

AN11

MASHRCRN

-

BPB

-

Anion front

FIG. 4. Comparison of molecular size dixiributions of Ch4-S from nasal sept,um and lamlwcy cartilage. Disc elcctrophoreais was carried out. in a modified buffer sptern, Tris-HCl-barbital at I)H 8.7. The ~xperimentnl c,onditions werc~ as dvwritwd in t,he text. A-E are the 1amprc-y 04-S contninin, u odd-numherctl pcl sliws. 1, 3. 5, 7 and 0; F is the combined extract, of the cvcn numbrwd ~(‘1 .sliws. (: is the combined c,strnct, of even-numhercd gel slices containing Ch4-S from nasal septum: H-I, arp the individual rstl,act of odd-numbrwd $liws 3. 5. 7. 9 and 11.

reservoir buffer was 0.03 M Tris-HCl, pH 8.2. After the electrophoresis the gel slab containing Ch4-S from lamprey cartilage was sliced at 2 mm intervals starting from the buffer front, and that of nasal septum, from the BPB dye front. The odd-numbered slices were extracted separately, and the even-numbered slices were combined, respectively. The gels were extracted according to the previous procedures. The extracts were analyzed by the same buffer system. The results al’e shown in Fig. 4, where the lamprey Ch4-S was also separated according to its molecular size. It is notable that the two leading bands were in front of the BPB band. Other buffer systems tested which would complete the resolution of the lamprey Ch4-S were Tris-HCl-glycylglycinc system using 0.05 M Tris-HCl, pH 8.2, for separating gel; ethanolamine-HCl-boric acid system using 0.05 M ethanolamine-HCl, pH 9.8. The buffers for the spacer gel became less critical as sulfated glycosaminoglpcans at such pH’s had elcctromobilities higher than that of the trailing anions. DISCUSSION

The experimental results clearly proved the observations that the diffuse bands obtained from acrylamide gel electrophoresis were due to size polpdispersity of the glycosaminoglycan chains. Since the diffuse

ELECTROPHORESIS

OF

GLI-COSAnIlSO(:L~CANS

391

band of Ch4-S was further subdivided into many fractions as described in this paper, the molecular weight determined by Hilborn and Anastassiadis (a), and Mathews and Decker (3) probably gave only an average weight of a mixture of widely and continuously distributed molecular sizes. It should be noted that, the method of purification nould tend to concentrate a fraction of the same charge density. Consequently, the present study does not discount the possibility that, glycosaminoglycan chains of different sulfate content have been eliminated in minor fractions. Numerous examples are available of variations in sulfate content, the most notable being the undersulfated chondroitin of cornea (13) and the oversulfated shark glycosaminoglpcans (14). Mobilities of sulfated glycosaminoglycans arc quite high; therefore, the discontinuous buffer systems generally used for disc clectrophorc& of prot’eins are not suitable for their analysis. The experimental results coincided with those observed by Richards and Gratzer (15) : RKA having a higher mobility in gel electrophorceis than that of a weakacid anion tends to travel as a single zone at the anion front, For the electrophorctic separation of sulfated glycosnminoglycans in acrylamide gel, a trailing anion and the running pH should be properly selected based on t’he elcctrophoretic mobilitics of glycosaminoglycans in the gel, which are regulated by t)he frictional resistance of the gel matrix and. therefore, depend on the molecular size. For disc electrophoresis of sulfated glycosaminoglycans, the first l)rcrequisite at the start of electrophoresis is t*hat the effective mobility of the trailing anion in the spacer gel must be lower than that of glycosaminoglycans in order to achieve the concentration of samples into an extremely thin starting zone; secondly, after the zone enters the separating gel, the effective mobility of the trailing anion must be faster than that of glycosaminoglycans, so that the trailing anion will run over the glycosaminoglycans and leave a uniform linear voltage gradient behind the moving boundary of the leading and trailing anions. The glycosaminoglycans left behind the boundary will migrate according to their mobilitics and the sieving cffcct of the gel. Since the niohilities of sulfated glycosaminoglycans arc high and inde!~cndrnt of pH above 6, the first prerequisit#e can he easily met by adjusting the pH of the spacer gel to a value below the I$, of the weak acid. This was evident from the experimental results, that the spacer gel became less important when samples were applictl in a thin layclr, hccnuse only a Plight rlifference in their mohilities was required to satisfy tlrc purpose. The second prerequisite is nrcomplishcd by raising the pH of the separating gel higher than the pKO of the n-cak acid to increase the effective mobility of the trailing anion over that of the glycosaminoglyrans t,o 1~ studied.

392

HSU,

HOFFMAN,

AND

MASHBURN

According to the experimental results, if the second prerequisite was not met, regulating the mobilities of glycosaminoglycans by the gel pore size was less effective. The Ch4-S from nasal septum was not completely resolved until the pH of the separating gel in t,he Tris-HCl-barbital system was raised to pH 8.2 (running pH, 8.6). The effective mobility of barbital at this pH is - 8.2 units (1 mobility unit = lo-” Cm’/V/sec) , indicating that Ch4-S from nasal septum has an electrophoretic mobility in the gel below -8.2 units. Therefore, for the separation of this Ch4-S, the pH of 8.2 for the buffer system was a correct, choice. Similarly, Ch4-S from lamprey cartilage has an electrophoretic mobility in the gel above -9 units. The mobility of diethyl barbiturate is - 10.2 units, therefore, in order to bring its effective mobility to above -9 units, the pH of the separating gel should be raised to the point where more than 90% of barbital was dissociated. In order to resolve these molecules of low molecular size but with high mobilit,ies, one would expect to need a trailing anion of high mobility, such as glycinate (-15.5 units), in order to make it easier to raise the pH of the separating gel. However, because glycine has a high pK,, the electrophoresis should be run at high pH. To avoid this, glycylglycine, with a low $7, and with a high electrophoretic mobility as an anion was tried. It gave the predicted result,. However, it was not used routinely because it is rather expensive. The general guide for choosing the pH and buffer syst.em for the disc electrophoresis is, therefore, summarized as follows: 1. Leading anion. Select an anion which has higher mobility than that of all other anions. 2. Trailing anion. Select a weak acid or an amino acid which has an effect’ive mobility less than that, of glycosaminoglycans at pH values and higher, at pH values higher than the pk-,. The lower than the pK, ; pH of the separat’ing gel is then set higher than the pK,,. 3. Counter ion. Use a weak base which has a 1X, as close to the pk’,, of t.he weak acid as possible, so that it, has buffering capacity at the pH’s of separating and spacer gel for both t.he st.rong and weak acids selected for leading and trailing anions, respectively. The pH of the spacer gel is set below the p& of the weak acid and as low as possible within the limits of hufferin g capacity of the base to ensure t,hnt the effective mobility of the trailing anion is sufficiently lower than that of glycosnminoglycans to achieve the best stacking. The distribution of Ch4-8 on disc clectrophoresis is due to the diffcrences in the length of polysarcharide chains, since both l)apain- mid alkali-trcatctl samples gnrc similar electrophorctir patterns. Singlr clion-

ELECTROPHORESIS

OF

GLTCOSAMIWOGLYCAKS

393

droitin sulfate chains with peptide were reported to be liberated from the tissue by digestion with papain, and polysaccharide chains essentially free of peptide may be obtained by treatment with alkali (9,16-18). It is not likely that the molecular weight difference is due to the size of the attached peptide, or to a number of polyeaccharicle chains attached to a single peptide. This is evident from the fact that the fractions having slow mobilities from a papain-treated sample did not change their mobilities on treatment with alkali. Also, the very low amino acid levels in papain- or alkali-treated chonclroitin sulfates would appear to preclude influences of peptides on mobility. Ch4-S isolated from bovine nasal septum by papain treatment was fractionat8ed into six fractions by gel chromatography on G-200 Sephadex by Wasteson (19). The molecular \Teights, determined by various methods were in the range of 10,000 to 40,000. There was, however, no confirmatory evidence in this case that peptidc digestion was complete, and it cannot be certain that only single polgsaccharide chains were involved in the large range of molecular n-eights. It’ is quite apparent from the magnituclc of separat’ions achieved by MathclTs and Decker (3) in differentiating single-chain polysaccharides from dimers that the separations described here involve differences of the order of dissacharides repeating units. Consequently, this method promises to be the most sensitive for a direct comparison of small difference; in molecular weight. Preparative methods are being developed to obt’ain sufficient amounts of fractions for adequate characterization and to serve as standards for calibration REFERENCES 1. Hsr, D.. HoFFnf.4s. P.. WI) M~SIIHCHS. T. .\.. JR. (1972) And. Biochem. 46, 156. 2. HILBORS. J. c.. ASD AS.~STASSIADIS. P. A. (1971) Awl. Bioch~m. 39, SS. 3. ~V~THEWS. iW. B., AND DECKER, L. (1971) Biochim. Biophys. Actn 244, 30. 4. POULIK. M. D. (1957) Nuke (London) 180, 1477. 5. ORSSTEIN, L.. .4x1 DAVIS. B. (1962) Disc Electl~ol)lloresis. Part I and II. Distillation Industries. Rochester, Kew Yolk. 6. HJERT~. S.. STVRE. J.. AND TISEI.II~S. A. (1965) Aurrl. Biorhenr. 11, 219. 7. ORNSTEIS. 1,. (1964) Atlu. IV. I*. Acrid. Sri. 121, 321. S. R~.~sHB~-RS. T. -4.. JR., .ZND HOFFMAX, P. (1966) And. Bioch~m 16, 267. 9. .~snmson-. B.. HOFFMAN. P., ISD MEYER. I<. (1965) J. Biol. (Ihem. 240. 156. 10. PESO. S., MFXIXR, I<,, .~~J)ERSON, IS.. AND HOFFMIX. P. (1965) J. Rio!. Chrm 240, 1005. 11. X~SHRURS. T. A.. JR.. ASD HOFFMAK. P. (1970) Auol. Biochem. 36, 213. 12. MATIIEWS. M. (1971) Biochem. J. 125, 37. 13. D.\vm~r~s, E. A.. AND METER. Ii. (1954) J. Biol. (‘hem. 211, 605. 14. Sr-KKI. S. (1960) J. Biol. Chem. 235, 3580. 15. RICII~I~DS. E. G.. .4311 GR.ITZRR. TV. R. (1968) iu C~r~omato~rxl)llic xnd Electro-

394

16. 17. 18. 19.

HSU,

HOFF;\IAN,

AND

MASHBURN

phoretic Techniques, Vol. II, Zone Electrophoresis (I. Smith, Interscience, New York. HOFFMAN, P., MASHBURN, T. A.: JR., MEYER, Ii., AND BRAY, 13. A. Chem. 242, 3799. LUGCOMBE, M., AND PHELPS, C. F. (1967) Biochem. J. 103, 103. MATHEWS, M. B. (1968) Fed. PWC. 27, 529. WASTESON. -1. (1971) Biochem. J. 122, 477.

ed.), (1967)

p.

419

J. L&2.