CHROMATOGRAPHY OF GLYCOGENS ON AGAROSE GELS

J. J. X~KSHALL’

Depavtnmzt of Biochemistry. University of Miami School of &ledicirw, Miami, Fla. 33130 (U.S.A.)

(Kcccivcd Xovcmbcr 6th. 1972)

The chromatographic behavior of several glycogens on the agarose gels Sepharose 2U and Sepharose 4B has been investigated. These are shown to be convenient media for examination of the polydispersity and relative molecular weights of different glycogens. Sepharose 2R is useful for the separation of artificial mixkres of glycogens and the components of glycogens occurring naturally as mistures of distinct popuiations of molecules.

INTRODUCTIOX

Gel chmmatc?grap!l) on devtran (Sephadex) ar?d polyacrylamide (Bio-Gel) gels has been widely used for the separation of proteins, nucleic acids and certain

carbc I! ‘:-ates on the basis of molecular weight I. However, the extremely high molel / ITIT- weights of polysaccharides, such as glycogens, rendered them non-sus- ceptible to fractionation by this technique since they are excluded from all grades of Bio-Gel and Sephadex. The recent development and commercial ava!lability of agarose gels of low degree of cros+linking has resulted in successful fractionation

of other high-molecular-weight polysaccharides, e.g. plant gums2, bacterial lipo- polysaccharide9 and certain mucopolysaccharides’+5. In this publication the use

of 2 y0 and 4 y! agarose gels (Sepharose 2B and 4B) for column chromatography of a number of glycogen samples from various sources is described.

EXPEHIMEKTAL

Materials Rabbit-!iver glycogen was extracted from the livers of both well-fed and

two-days starved animals using the mercuric chloride procedure of MORDOH et al?. After purification by three reprecipitations, tollowed by dialysis and treatment with mixed-bed ion-exchacge resin (Bio-Rad Laboratories) to remove residual mercuric chloride, the polyza, ,narides were isolated by freeze-drying. Normal human-liver glycogen was the sanlple examined by MERCIEK AND WHELAN?. Human- macle glycogen, prepared by the KOH procedurti, was supplied by Prof. D. J. MANNERS. Glycogen from the muscle of Type II g!ycogen storage disease patients

*Correspondence to: Dr. J. J. MARSHALL, Department of Biochemistry, University of :,xiarni S;hoo! gr “f+-:..” 1) (1 la.-.” P-c ~;-.--xmr. bnnrv _&fiap$, F!?.. z?I~, II S.A. . ., cu C.,,,,) 1 .V. _I._ “,J, _..,_ “,” - ____-_ -..,

202 j. J. MARSHALL

was prepared by cold-water estraction of the tissue and kindly provided by Prof. BRESM E. R~xAs. _-Iscu~is and tape-worn) glycogcns were donated by Dr. S. A. ORRELL. Shellfish glycogen was : .irchased from Nann Research Laboratories and was three times reprecipitated b *rare use. Phytoglycogen A was prepared from immature sweet corn as by PEAT et u.I.~. Unfractionated sweet-corn grycogen was prepared by homcgenization of sweet corn kernels in mercuric chloride solution as in the initial stage of phytogiycogen A preparations. After centrifugatior to remove grain residues the solution was dialyzed, then treated with mixed-bed ion- exchange resin. So attempt ;vas made to isolate the polysaccharidc from the solution, this being subjected directly to chromatography.

!3‘ (bl

CHllOMATOGliAPHY 01; trL\‘COGEXS 203

Sepharose 2B (Lot 50-C-0630), Sepharose 4B (Lot 30-C-1560-1) and Blue Dextran 2000 were products of Pharmacia Fine Chemicals. Glucoamylase from Aspergillus n&*v was prepared by QURESHI~O, using the method of FLEMIXG AND STONI+. Glucose osidase (Grade 11) and horseradish peroxidase were obtained from Boehringer Mannheim GmbH.

Moleczdar-siere claronaatograplzy Chromatography was performed in columns of dimensions 90 x 1.5 cm wiih

gel beds approximately 57.5 cm high. Elution was with I 9& sodium chloride solution under a head of 12-15 cm of elwnt or with an LKB Varioperpex peristaltic pump, the flow-rate being approximately j ml/h. Under the conditions used no variation in the bed volume took place during the period of several weeks continuous use.

Samples containing polysaccharide (approsimately IO mg) dissolved in eluent (I ml) were applied to the top of the gel bed and fractions of constant volume (2.5-3.5 ml) collected auton\atically. Concentrations of giycogen in column fractions were determined enzymically by quantitative conversion into glucose using Asper- gilltfs fziger glucoamylase and a-amyiase It, followed by measurement of glucose

l’ig. I. Distribution of carbohydrate in the cttlucnt from R column of Scpharose zk3 after chromatography of: (a] fed rabbit-liver glycogcn; (b) starved rabbit-liver glycogen; (c) shcll- fish giycogcn; (d) human-liver glycogen; Ic) human-muscle gly.cogon; (f) Type II glycogenosis muscle glycogcn; \g) phytoglycogcn A; (h) sweet-corn juice: (1) Blue Dextran + glucose. For experimental details see the test. For convenicncc, carbohydrate concentrations have been shown in purely relative terms.

20-t J. J. MARSHALL

with glucose osidase as by LLOYD AXD \'VHELAN~~. Blue Dextran was detected by measurement of the absorption of fractions at Goo nm.

Analytical ultracentrifugation was carried out in a Spinco Model E ultracen- trifuge at speeds of the order of ~o.ooo r.p.m.

RE’XLrS

Figs. Ia-h show the results obtained on chromatography of individual gly:ogen samples on Sepharose zB. Fig. xi shop the distribution of Blue Dextran from the same column and the elution of glucose (total column volume). The result:; of chromatographr of shellfkh o_ +cogen, phvto$vcogen, Blue Dextran ” 4 and glucose on Sephhrose qE are given in Figs. ?a-c. Fig, 3 illustrates the results

Fix. L. Distribution of carhohpirate 13 the dluent from a column of Sq~harosc +I3 after chromato&Taphy of: (a, shellfish +w~en : 1 tjr phytoklycogcn .\ ; I.CI 131~ Lwstran -. ~lucosc.

of successful fractionation experiments using Sepharose 2B; Fig. 3a shows the separation of a mixture of approximately equal amounts of (fed) rabbit-liver glycogen and shellfish glycogen: Figs. 3b and 3c show the separation of the high- and low- molecular-weight compone& of .4scuris and tape-worm glycogens on the same

CHROMATOGRAPHY OF GLSCOGEKS

Fig. 3, ‘/I ation of Rl?cojicn-components using Scpharosc 2B: (a) misturc of fed rabbit-liver giycogcn and shellfish glycogcn; (b) Ascaris gl~co~cn: (c) tape-worm glycogen

Fig. 4. Sedimentation of: (a) ilscavis; and (b) tape-worm glycogens in the analytical ultra- centrifuge. The photographs wre taken 1 L min after reaching maximum speed (9.341 r.p.m.). Sedimentation is from left to right. (Courtesy of Dr. S. A. OKRELL.)

column. The sedimentation behavior of Ascaris and tape-wo-m giycogens in the analytical ultracentrifuge are shown in Figs. qa and 4b

CHROW.ToGRAPHY OF GLYCOGESS 207

and polydisptxsit~~. Sepharose 4B may also be of use for this purpose, although its use will be restricted to the lower-molecular-weight glycogens. Even then, it does not appear to offer any m ajar adxmtagcs over Sepharose 213. Thus, chromato- graphy of phytoglycogen A and shellfish glycogen (Figs. 2a and 2b) still does not reveal any inhomogeneity in these glycogens. In the latter case particularly this is of some significance. One of the reasons for the present work was to discover whether the macrodextrins left after treatment of this glycogen with a-amylse2fJ-28

do arise from shellfish glycogen molecules per se, or whether they exist as a separate population of molecules whose presc?nce only becomes detectable after :t-amylase action. Our results would, however, appear to be inconsistent with this lakter idea and we must therefore accept that macrodestrins do arise from highly branched regions of the shellfish glycogen molecules as suggested by SCHRAZIM and co- workers20-23.

From Figs. Ia and IC it is clear ti&, i? the absence of any intermolecu:.:r interactions, a mixture of rabbit-liver and shellfish glycogens should be largely separated by chromatography on Sepharose 2B. This was found to be the case when these glycogens were co-chromatographed (Fig. 3a), the resultant carbohydrate elution profile being what would be expected on the basis of Figs. Ia and IC. In the past the standard technique for examining the heterogeneity of glycogens has been analytical ultracentrifugation 17. BUBDIXC and his co-workers have in this way esamined a large number of glycogec sampics prepared m different ways and from different som-ceP* l% “49 25. In some cases clear indicatix of populations of molecules of two different sizes has been obtained (Figs. + and 4b) and these have

been part l!. ,l)arated by zonal ult.raccntrifugation’J. Chromato,~aphy of such samples 01\ , illarose 213 also results in separation of the two component: II%. 11~ and SC). It is anticipated that, in view of its facility, the present technique will lie useful for fractionation r;f ~nulticomponent glycogens on a preparative scale, thus allowing a detailed comparison of the constituent fractions.

It is not possible at the present time to use the method described for the determination of absolute molecular weights, in view of the absence of suitable standards toi- calibration purposes. This possibility does, however, exist. The distri- bution of Blue Dextran 2000, a colored polysaccharide with a reported average molecular weight of 2. IO*, is shown on columns of both types but it is likely that the elution volume of this near-linear polvsaccharide will correspond to that of a branched polvsaccharide of much higher molecular weight, in view of the much more compact nature of the glycogen molecule. Some idea of the mean molecular

weights of the ~!~co~Cns chromatographed ~a>-, iIu\vcver, be ubtaiued by using

a value of x9--20- IO” for that of sweet-corn glycogen2h. On this basis it is difficult to see the molecuIar weights of the two components of .Qscavis glycogen being 450 and so* 100 as suggested by ORREL et r~l.~~.

Further uses of this techinque are for the examination of the mechanism of action of glvcogen synthesizing and degrading enzyn,?s. T:IUS esamiilation of the carbohydrate eiution profiles after different cstents of h+olysis may be Useful

in determining the action pattern (i.e. endo or eso attack) of debranching enzymes such as isoam:.!ase27*2R on glycogea. It should also be a convenient method for detect-

ing whrlthzr higher- or lower-molecular-weight molecules are preferentially Utilized as substrates for various enzymes such as glycogen syntletase and phosplwylase20.

20s .j. j. XARSH‘UL

Additionally it is likely to be useful for examining glycog~,~s from organisms subjected to different metabolic stresses, and particularly for stud_ying the diitri- bution of radioactive label in glycogen molecules of different sites during metabolic studies. The application of the method to the separation of the components of various starches will be described elsewherew.

ACKXOWLEDGEHESTS

This work was supported in part by a grant from the National Science Foun- dation (GB 27491). I thank Professor I\:. J \vHELAN for his interest and helpful

discussion.

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