PRODUCTION OF HEPARINASE BY BACTEROIDES1

BERTRAM M. GESNER2 AND CHARLES R. JENKIN3Department of Medicine, New York University College of Medicine, New York,, New York

Received for publication September 27, 1960

To date, the only bacterial heparinase whichhas been extensively studied was derived froma Flavobacterium isolated from soil (Payza andKorn, 1956; Korn and Payza, 1956; Korn, 1957).Despite the close chemical similarity of heparinand chondroitin sulfate, enzymes derived fromProteus vulgaris which have been shown to de-grade chondroitin sulfate have been found to beinactive against heparin (Martinez, Wolfe, andNakada, 1959). During studies of various bio-logical effects of heparin in this laboratory, it wasfound that the normal intestinal flora in man con-tained as a predominant organism a bacteriumcapable of producing enzymes which degradedseveral mucopolysaccharides, including heparin.The active bacterium, a strict anaerobe, was

identified as a species of Bacteroides. Because ofits poor growth on ordinary media and becauseof the necessity of obtaining relatively largequantities of bacteria for enzyme extraction, asearch for growth-enhancing factors was made.The development of a medium in which growthof the organism was accelerated made morefeasible a study of some chemical and biologicalproperties of a partially purified cell-free extractpossessing heparinase activity. The results ofthese studies are the subject of this report.

MATERIALS AND METHODS

Materials. Sterile aqueous catalase (1,000units/ml) was obtained from Mann ResearchLaboratories, Inc. Toluidine blue (total dyecontent 73%) was purchased from the AlliedChemical Dye Corporation. Alumina A-301C wasobtained from the Aluminum Company ofAmerica. 3,5-Dinitrosalicylic acid for reducingsugar assay was obtained from Eastman KodakCompany. For investigating the effect of pH

1 This investigation was supported by USPHStrainee grant 2E-S.

2 Present address: Sir William Dunn School ofPathology, Oxford University, England.

3 Present address: Department of MicrobiologyAdelaide University, South Australia.

on the degradation of heparin by the partiallypurified fraction, the following buffer systemswere used: citric acid-sodium citrate pH 4.0,5.0, 5.5, 6.0; monobasic sodium phosphate-dibasicsodium phosphate pH 6.0, 6.5, 7.0, 7.5, 8.0;sodium barbital-hydrochloric acid pH 7.5, 8.0,8.5, 9.0; all at 0.025 M. Since preliminary evidencesuggested that the presence of magnesium ionmaintained the activity of the extracts, 0.2 gmagnesium chloride was added to each liter ofbuffer.

Substrates. Sodium heparin and sodium chon-droitin sulfate were purchased from Mann Re-search Laboratories, Inc. Potassium hyaluronatewas kindly supplied by Maxwell Schubert andAlan Johnson of the Department of Medicine,New York University College of Medicine. Apartially purified mixture of blood group sub-stances A and B was obtained from Merck,Sharp and Dohme. Purified Escherichia colilipopolysaceharide was prepared by the methodof Westphal, Lilderitz, and Bister (1952).

Assay procedure. The disappearance of heparinwas followed by the decrease in metachromasiawith toluidine blue using a modification of themethod of Jaques, Mitford, and Ricker (1947).An 0.02% aqueous solution of toluidine blue wastitrated against samples of reaction mixturescontaining 0.1 to 10.0% heparin. During thetitration, the dye was converted to a red colorand a flocculant blue precipitate formed. Whenalmost all the heparin had been titrated, thereaction mixture became colorless. The additionof 0.1 to 0.15 ml of dye then colored the reactionmixture blue and this was taken as the end point.This assay does not necessarily measure intactsubstrate since partially degraded heparin maystill show a metachromatic reaction.Reducing groups were estimated after the

method of Meyer and Gibbons (1951) in which 0.5ml of the test samples was subjected to 0.5 ml of1.5% 3,5-dinitrosalicylic acid and 0.5 ml of 6 MNaOH for 30 min at 60 C. The mixture wasdiluted with 10 ml of water and optical density

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was read at 510 mA using maltose as the standard.Since the breakdown products of heparin wereunknown, the amount of reducing groups re-ported represent only the relative reducing valuepresent in a test sample as compared to thearbitrarily chosen standard, maltose. The valuesdo not represent an exact determination ofmicromoles of any specific reducing substanceliberated from the substrate.Hexosamine (or hexosamine end groups) were

determined by the Elson and Morgan (1933)reaction using glucosamine as the standard andreading at 530 m,u. Catalase was assayed bythe method of Herbert (1955). Protein contentof the fractions obtained by ammonium sulfatefractionation was estimated by the Folin andCiocalteau (1927) method using bovine albuminas a standard.

Media. Two basic types of media were usedfor the isolation and study of growth characteris-tics of the Bacteroides: (i) Tryptose phosphatebroth (Difco). (ii) Fluid thioglycolate medium(Difco).For growth of large quantities of Bacteroides,

a medium containing the following ingredientswas used: trypticase, 15 g; yeast extract, 5 g;glucose, 2 g; sodium chloride, 2.5 g; sodiumthioglycolate, 0.5 g; 1-cysteine, 0.5 g; methyleneblue, 0.002 g; water, 1 liter. This medium issimilar to thioglycolate broth, but contains noagar as this interfered with the harvesting of thebacteria. Since it was found that filtrates of E. coliwhich contained catalase enhanced the growthof Bacteroides, each liter of medium was madeup to contain 100 ml of sterile filtrate of E. coli.Similar results could be obtained by adding10,000 units of catalase per liter of medium, butthe former was used routinely because of theexpense of large quantities of sterile aqueouscatalase.

Preparation of Bacteroides for enzyme extraction.Preliminary experiments showed that extractsprepared from Bacteroides grown in the absenceof heparin had no detectable heparinase activity.For the preparation of an active extract, sodiumheparin was added to the medium to give aconcentration of 0.1%,. The inoculum was grownovernight in heparin-containing medium andadded in sufficient amount to provide an initialconcentration of 106 bacteria/ml. The mediumwas distributed in Erlenmeyer flasks so thatlittle air space remained between the surface of

the medium and the cotton wool plug, to main-tain anaerobic conditions. The cultures wereincubated at 37 C for 48 hr, at which time about30 to 50% of the heparin had been degraded asindicated by the decrease of metachromasia. Thebacteria were harvested by passing through aSharples centrifuge at 4 C and frozen at -20 C.

Preparation of crude enzyme extract. Using themethod of Mcllwain (1948), 5-g samples of thefrozen bacteria were ground with 12.5 g ofalumina for 5 min at 4 C to form a thick paste.The mixture was suspended in 30 ml of 0.025 Mphosphate buffer at pH 8.0 and gently swirledon a rotary shaker for 60 min at 4 C. After this,the suspension was centrifuged at 15,000 rev/minfor 30 min at 4 C and the resulting supernatantfluid used as the crude extract. Extracts ofBacteroides grown in the absence of heparinwere similarly prepared.

Partial purification of the extract. Ammoniumsulfate fractionation of the crude extract wascarried out at 4 C. The resulting fractions wereall assayed for their heparinase activity afterdialysis overnight against 0.025 M phosphatebuffer, pH 8.0 at 4 C.

Five milligrams of heparin in 0.1 ml of 0.025 Mphosphate buffer at pH 8.0 were added to 2-mlsamples of the fractions. The mixture was incu-bated at 37 C for 2 hr and assayed for the releaseof reducing substances.

RESULTS

Isolation of a bacterium capable of degradingheparin. Normal human stool samples (approxi-mately 1 g) were suspended in 10 ml of salineand 1 ml of this suspension was used to inoculate10 ml of tryptose phosphate broth containingheparin. The cultures were incubated at 37 Cand samples were taken for heparin assay at24-hr intervals. In every case of more than 50different samples of stools tested, all the titratableheparin disappeared within a few days. A typicalrate of degradation by a crude stool sample isshown in Fig. 1. Heparin degradation was foundin dilutions of at least 10-6 in all of the crudestool samples tested. In samples of uninoculatedmedia or in samples inoculated with variousbacteria, no decrease in metachromasia wasobserved.

Serial dilutions of several stool samples weremade in tryptose broth and samples of thehighest dilution (usually 10-7 to 10-9) which were

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6*

ah.

E 2

O 1

4

E24

0 2 3 4 5 6 7

Days

Fig. 1. Degradation of heparin by bacteriaderived from human stool. In all of these experi-ments the bacteria were inoculated into 10 ml oftryptose broth containing heparin. All of thecultures were grown aerobically at 37 C exceptfor the sample containing Bacteroides alone whichwas incubated at 37 C in a Brewer anaerobe jarcharged with hydrogen. In the samples wherepure cultures were used the inoculum was approx-

imately 107 bacteria.

found capable of degrading heparin were platedonto chocolate blood agar. The plates were thenincubated either aerobically, or anaerobically in a

Brewer anaerobe jar charged with hydrogen. Theaerobicallygrown plates appeared to consist ofa pure growth of E. coli. No single colony isolatesfrom these plates were capable of degradingheparin when grown either aerobically or

ianaerobically in tryptose broth containing thissubstrate (Fig. 1). However, under anaerobicconditions, there appeared, after 3 to 5 daysincubation at 37 C, small, pinpoint coloniesimmediately adjacent to the large colonies ofE. coli. These small colonies were found toconsist of gram-negative, pleomorphic bacteria,varying in shape from small rods to long fila-ments. They were nonmotile, had rounded ends,and were nonsporeformers. Bacteria having these

characteristics were found in every instance(5 separate stool samples) where the isolationof the heparin-degrading organisms was carriedout. The bacteria were tentatively identified asmembers of the genus Bacteroides. It is note-worthy, however, that the ability to degradeheparin may not be a ubiquitous characteristicin this genus since preliminary experimentssuggested that Bacteroides tumidus and Bac-teroides limosus (obtained from the AmericanType Culture Collection) did not cleave thismucopolysaccharide.

Single colony isolates of the Bacteroides grewpoorly in tryptose broth in the anaerobic jarand in fluid thioglycolate medium in the absenceof E. coli, and not at all aerobically. Tubescontaining an inoculum of 103 to 104 bacteria/mldid not show growth even under anaerobicconditions. However, larger inocula (106 bacteria/ml), when grown anaerobically in the heparin-containing fluid media, were found capable ofdegrading this substance as measured by thedecrease in metachromasia (Fig. 1). When amixture of E. coli and Bacteroides was used,much more rapid degradation of the heparintook place (Fig. 1) even when culturedaerobically.

Growth-promoting factors for Bacteroides. Thefindings that (i) pure cultures of Bacteroidesunder anaerobic conditions grew poorly anddegraded heparin relatively slowly, (ii)Bacteroides grown with E. coli aerobicallydegraded heparin relatively rapidly, and (iii) E.coli alone did not degrade heparin, suggestedthat the E. coli was providing growth-promotingfactors for the Bacteroides.To investigate the role of E. coli in the mixed

cultures, the following experiments were per-formed. A 48-hr growth of E. coli cultured intryptose broth was centrifuged at 7,500 rev/minfor 20 min at 4 C and the supernatant filteredthrough a sintered glass bacterial filter. One-milliliter samples of the sterile filtrate wereadded to 10 ml of tryptose broth or thioglycolatemedium and the medium inoculated with ap-proximately 107 Bacteroides. No growth occurredin the tryptose broth incubated aerobically.However, in fluid thioglycolate medium, and intryptose phosphate broth incubated anaerobi-cally, growth was markedly enhanced; concen-trations up to 5 X 108 bacteria/ml being obtainedafter 24 hr incubation at 37 C.

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Since it had been suggested by Beveridge(1934) that E. coli may enhance the growth ofBacteroides necrophorus, by providing catalase,sterile E. coli filtrates were tested for this sub-stance by the method of Herbert (1955). Thesterile filtrates were always found to containcatalase activity although the concentrationvaried for several preparations. The catalaseactivity was destroyed by autoclaving the filtratebut this procedure did not entirely eliminate thegrowth-enhancing effect of the filtrate.To test the effect of catalase alone on the

0.8-

0.4-

00.2

0 20 40 60 80 1OOUnits of Catalog. / ml medium

Fig. 2. Effect of varying concentrations ofcatalase on the growth of Bacteroides related toheparin degradation. Ten milliliters of tryptosebroth which contained known amounts of catalasewere inoculated with the same quantity of Bac-teroides (approximately 107) incubated anaero-bically at 37 C for 48 hr. The lower line indicatesthe per cent decrease in metachromasia.

growth of Bacteroides, various concentrationsof sterile aqueous catalase were added to 10-mlsamples of tryptose broth and each tube in-oculated with the same quantity of Bacteroides(approximately 10 bacteria). No growth occurredin the samples incubated aerobically. However,under anaerobic conditions, catalase markedlyenhanced growth. The degradation of heparinwas proportional to the amount of growth(Fig. 2). The same growth-promoting effect wasobserved in thioglycolate medium.The finding that catalase was effective in

promoting growth only under anaerobic condi-tions suggested that the E. coli might also beproviding an anaerobic environment. To testthis point, methylene blue was added to tryptosebroth (to serve as an Eh indicator) and 10 mlof medium inoculated with a colony of E. coli.After 8 to 16 hr incubation at 37 C, the dye wascompletely decolorized, indicating that an-aerobiosis as well as catalase were, in fact,provided by the E. coli.

Degradation of heparin by a cell-free, partiallypurified fraction of Bacteroides. Utilizing thenewly designed media, sufficient quantities ofBacteroides were obtained for the preparationof partially purified, cell-free extracts whichcontained heparinase activity. Active extractswere prepared from Bacteroides obtained fromseveral separate stool samples. The extractsused in the experiments that follow, however,were derived from a single isolate.

Crude (Alumina) Extract*

Saturated (NH4)2SO4 at pH 7.5 added to 50%saturation. Centrifuge at 10,000 rev/minfor 20 min.

PrecipitateResuspend in phosphate buffer (0.025 M,pH 8.0). (FRACTION 1)

Supernatant FluidSaturated (NH4)2SO4 pH 7.5 added to 66%saturation. Centrifuge 10,000 rev/min,20 min.

Precipitate Supernatant FluidResuspend in phosphate (FRACTION 2)buffer (pH 8.0, 0.025 M).(FRACTION 3)

Fig. S. Ammonium sulfate fractionation of the crude extract*This procedure also prrvided active fractions from Bacteroides grown in the presence of Escherichia

coli on tryptose broth containing heparin. Extracts of E. coli similarly prepared did not possess heparin-ase activity.

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90

a 80

E° 70

Z 60E

50a

a., 40-a1

c 30

a. 20

l0o

0 60 120 180 240Minutes

Fig. 4. Degradation of heparin by partiallypurified cell-free extract of Bacteroides. Onemilliliter of the extract was incubated with 10 mgof heparin at 37 C, pH 7.8. The extract contained1.5 mg protein/ml. One-tenth milliliter samplesof the reaction mixture were used for assay.

Using the fractionation procedure outlined inFig. 3, 50 to 80% of the heparinase activity was

usually found to be associated with fraction 3.Partially purified fractions obtained by thisprocedure were used in the experiments whichfollow.The degradation of heparin was determined

by the decrease in metachromasia (Fig. 4) andthe release of reducing groups (Fig. 5). Thepartially purified fraction with no heparin addedand heparin in buffer were used as controls inall the experiments. Crude extracts preparedfrom Bacteroides grown on medium containingno heparin had no heparinase activity.The activity of the fractions diminished

slowly during storage for several weeks at 4 C.Preliminary studies indicated that the activity

of the extract could be abolished by dialysisagainst sodium versenate and that it could berestored with magnesium ion. However, furtherwork is needed to determine whether, as in the

Minutes

Fig. 5. Release of reducing groups from heparinby partially purified cell-free extract. Three mil-liters of the extract were incubated with 5 mg

of heparin at 37 C, pH 7.8. The extract contained1.5 mg protein/ml.

1.0

0

*Z 0.75_ _

E

7i

0.50-

0%

4 5 6 7 8 9

pH

Fig. 6. Effect of pH. One milliliter of extract(diluted 1:1 in respective buffer) was incubatedat 37 C for 2 hr with 5 mg of heparin. The extractcontained 1.5 mg protein/ml.

0

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2.5

E

2.0-

0 1.5a-

1.0

0

E

0.5

01 3 5 10 20

mg Heporin/ml

Fig. 7. Effect of varying concentrations of sub-strate. One milliliter of the extract was incubatedwith the indicated amount of heparin at 37 C,pH 7.8 for 2 hr. The extract contained 1.5 mg

protein/ml.

2.0-

0

I.5_*/_0~~~~E/

1.0 _

,0.5

E/

0 0.2 0.4 0.6 0.8 1.0

ml Extract

Fig. 8. Effect of varying concentrations ofenzyme. A total of 1 ml of reaction mixture con-

taining the amount of extract indicated was in-cubated with 5 mg heparin at 37 C pH 7.8 for 2hr. The extract contained 1.5 mg protein/ml.

Flavobacterium system, inhibition by versene

might be due to an effect other than chelation.Effect of pH and temperature on the degradation

of heparin. Two-milliliter samples of a 1:1 dilution

- 4.5 _ "_ '4.5 ;C E~~~~~~~~~~~.' / ~~~~~~~~~~EE 4.0oo4.O ~~~~~~~~~0o /

cp3-5 -_ 3.5 >

3.0 o - 3.0 EE 01

0L 2.5 - 2.5w0~~~~~~~~~~~~c. 2.0 - 2.0 c.C EU ~~~~~~~~~~~00.5 - 1.5 0

-1.0 i.nt0E 0

E0.5 0.5 ~

0 60 120 180Minutes

Fig. 9. Release of hexosamine or hexosamineend groups from heparin. Eight milliliters of theextract containing 10 mg of heparin/ml were in-cubated at 37 C, pH 7.8. Samples for hexosamineand reducing group assay were taken simultane-ously from the same reaction mixture. In thisexperiment the extract contained 3.2 mgprotein/ml.

of the extract in buffer were dialyzed overnightagainst 500 ml of the various buffers at 4 C.Following dialysis, 5 mg of heparin in 0.1 mlof the respective buffers were added to 1-mlsamples of the dialyzed extract. The mixturesof enzyme and substrate were incubated at 37 Cfor 2 hr and the degradation of heparin, asmeasured by the release of reducing substances,estimated at the end of that time. From theresults presented in Fig. 6 it is apl)parent thatoptimal activity was present over a fairly widepH range, i.e., pH 6.5 to pH 8.0. The experi-mental procedure was also a measure of the sta-bility of the enzyme(s) after dialysis overnight at4 C with the different buffers used. Where the pHwas the same for the different buffers, the activitywas similar.

Three-milliliter samples of the active fractionwere incubated with 5 mg of heparin at 4, 21,37, and 42 C. No enzyme activity could bedemonstrated at 4 C, whereas considerabledegradation occurred between 21 and 42 C;optimal breakdown occurred at 37 C. The

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activity was completely abolished by heatingat 60 C for 30 min and by boiling for 10 min.

Effect of varying concentrations of substrateand enzyme. As is shown in Fig. 7 the amountof reducing groups released was approximatelyproportional to the concentration of heparinover a range of about 1 to 5 mg/ml. The releaseof reducing groups was found to be proportionalto the enzyme concentration in 1 ml of reactionmixture (Fig. 8).

Release of hexosamine from heparin. The releaseof reducing groups from heparin could resultfrom the splitting of a variety of chemical bondspresent in this polysaccharide. Similarly, a

decrease in metachromasia could be explainedby more than one specific type of enzyme activity.To better define this heparinase enzyme system,the release of hexosamine end groups fromheparin was determined.

Total reducing groups and hexosamine endgroups were assayed simultaneously usingglucosamine as the standard for both determina-tions. It wNill be noted in Fig. 9 that the valuesfor total reducing groups are several times greater

1.25

ol

E

._

-i

'.

1.0

0.75

0.5

0.25

0 30 60 90

Minutes120

Fig. 10. Degradation of other mucopolysac-charides. Four milliliters of the extract (diluted1:1) were incubated at 37 C, pH 7.8 with 10 mgof substrate. The extract contained 4.6 mg pro-tein/ml.

C hondrotI tin

E°1.25 SSu fat

° 1.02o/5E

.0

1.0

0 0.75_y/ ont

0 50

0~~~~~~E 0.25

Minutes

Fig. 11. Degradation of mucopolysaccharidesby crude extracts derived from Bacteroides grownon medium containing no heparin. Three-millilitersamples of crude extract were incubated at 37 C,pH 7.8 with 10 mg of heparin or chondroitin sul-fate. Four milliliters of crude extract (diluted1:1) were incubated under the same conditionswith 10 mg hyaluronate.

than the hexosamine values. The interpretationof these results will be discussed.

Substrate specificity of extracts derived fromBacteroides grown in presence and absence ofheparin. The ability of the active fraction todegrade other naturally occurring mucopolysac-charides was determined by assaying the releaseof reducing substances from sodium chondroitinsulfate, potassium hyaluronate, a mixture ofblood group substances A and B, and lipopolysac-charide prepared from E. coli. The fraction was

found to be inactive against the latter two prep-arations. However, as shown in Fig. 10, sodiumchondroitin sulfate and potassium hyaluronate,as well as sodium heparin, were found to bedegraded.

Although crude extracts prepared fromBacteroides grown in the absence of heparinwere found to have no heparinase activity,they were capable of degrading potassiumhyaluronate and sodium chondroitin sulfate.This finding is shown in Fig. 11. Taking intoaccount the dilutions of extract and substrateused, the rates of hyaluronate and chondroitinsulfate degradation are similar.

Hyolurono to

Heporin

C hondroitinSulfate

/ I

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Biological activity of degraded heparin. Theanticoagulant activity of 1 mg of heparin wasdestroyed after incubation with 1 ml of the activeextract for 4 hr at 37 C. When the fraction wasfirst boiled for 10 min, or the extract from or-ganisms grown on the medium containing noheparin was used, the anticoagulant effect ofheparin was retained.

In preliminary experiments, lipoprotein lipasewas demonstrable in the serum of dogs 5 minafter the intravenous injection of 5 mg of heparin.This effect was eliminated if the heparin hadbeen incubated prior to injection with 2 mlof the active fraction at 37 C for 4 hr.

DISCUSSION

A bacterium isolated from human stool andidentified as a species of Bacteroides has beenshown capable of degrading heparin. As had beennoted for other members of this genus, thisorganism was found to grow poorly on ordinarymedia in pure culture. This problem and theinteresting relationship with E. coli prompted asearch for growth-promoting factors. The dataindicate that anaerobiosis is necessary for growth,and that catalase under these conditions enhancesgrowth.The data further suggest that E. coli per-mits growth under apparent aerobic conditionsby producing anaerobiasis in the medium,and enhances growth by providing growth-promoting factors. Since E. coli culture filtratescontained catalase activity, it is likely thatthis enzyme is one of the growth-promotingfactors provided by that organism. However,the fact that the growth-enhancing effect ofthe filtrate was not completely abolished byboiling suggests that other growth-promotingsubstances may be provided by the E. coli orthat the product of heat-inactivated catalasemay be utilized to advantage by the Bacteroides.The above evidence may be taken to correlatewith Beveridge's (1934), conclusions that thesurvival and proliferation of B. necrophorusdepends at least partially on the relative veloc-ities of (i) peroxide and catalase formation and(ii) oxidation and reduction, and that the forma-tion of catalase and oxygen absorption mustplay some part in the symbiotic effect of E. coli.Also, since catalase is a heme protein, it is perti-nent that in a recent report by Gibbons andMacDonald (1960) it was noted that severalstrains of Bacteroides melaninogenicus require

hemin for growth. These workers found thatsome of these strains also required a diffusablegrowth factor produced by Staphylococcus aureusand that vitamin K could effectively replace thegrowth factor.

Several species of Bacteroides have beenshown to degrade dextrans and related muco-polysaccharides (Sery and Hehre, 1956) but theability of this organism to degrade heparin hasnot been previously described. One other bacterialheparinase system which has been studied wasdescribed by Korn and Payza (1956). Theseworkers were able to induce heparin-splittingenzymes in a Flavobacterium isolated fromsoil. Crude acetone extracts prepared from thisbacterium degraded heparin, chondroitin sulfate,and hyaluronic acid. The enzyme system de-scribed in this paper closely resembles thatstudied by Korn and Payza.The evidence presented here suggests, as has

been pointed out for the Flavobacterium system,that several enzymes are responsible for thedegradation of heparin. If all of the aminogroups of heparin are bound to sulfur, then asulfamidase as well as a glycosidase must bepresent in the extract, since the Elson andMorgan reaction used to measure the release ofhexosamine end groups requires that both theamino and the aldehyde groups of the aminosugar be free. Further, if the constituents of thereaction mixture do not interfere with the Elsonand Morgan reaction, then the results wouldindicate that more reducing groups than hexosa-mine end groups are liberated. This could beexplained in various ways: for example, if morethan one type of glycosidase were present, or, ifmore glycosidic bonds than sulfamidic bondswere split on the hexosamine end group, thereducing group value could be greater than thehexosamine end group value.As has been noted for the Flavobacterium

system (Korn, 1957; Hoffman et al., 1957), ex-tracts prepared from organisms grown on hepa-rin-containing medium degraded chondroitin sul-fate, hyaluronate, and heparin; however, it wasnot necessary to add this substrate to the mediumto obtain extracts containing enzymes activeagainst chondroitin sulfate and hyaluronate. It ispossible, therefore, that this substrate causes theproduction of significant quantities of enzyme(s)which can degrade heparin to a stage whereenzymes ordinarily present in significant amount

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can act. A more precise characterization of theenzymes involved here requires further workalong the lines carried out for the FlavobacteriumSystem.The finding that Bacteroides, a common

anaerobe of the humain intestinal tract, possessesenzymes capable of degrading naturally occurringmucopolysaceharides may have some significancein relation to the pathogenesis of certain entericand connective tissue disorders. Further, it wouldseem desirable to look for the production ofmucopolysaecharidases in Bacteroides isolatedfrom human lesions, since these enzymes mayplay a role in the development of the regionalthrombophlebitis which is a characteristic featureof Bacteroides infection (Reid et al., 1945).

ACKNOWLEDGMENTS

The authors are most grateful to Chandler A.Stetson and William S. Tillett for their sulg-gestions, criticisms, and encouragement.

ADDENDUM

It is noteworthy that Mandel and Racker(1953) found that a mucopolysaccharide isolatedfrom mouse intestines was capable of inihibitingthe infectivity and hemagglutination of Theiler'sGDVII strain of encephalomyelitis virus. Themucopolysaccharide inhibitor was found to bedestroyed by enzyme(s) present in the feces ofmice and other animals. Subsequently (Mandel,1960, personal communication), it was found thata species of Bacteroides isolated from mouse in-testines was responsible for the production of themucopolysaccharidase (s).

SUMMARY

A species of Bacteroides, normally a predomi-nant organism in human stool, has been isolatedand found to degrade heparin. In pure culturesand on ordinary media, the organism growspoorly. However, growth of this strict anaerobecan be enhanced by catalase or sterile filtrates ofEscherichia coli.An extract prepared from Bacteroides grown

on a heparin-containing medium possessedenzymes capable of degrading this substrate andrelated mucopolysaecharides. Partial purificationof these enzymes was accomplished by ammoniumsulfate fractionation. Some of the chemical andbiological properties of the heparinase systemhave been described and the results suggest that

several enzymes are involved in the cleavage ofheparin.

Extracts from organisms grown in the absenceof heparin had no detectable heparinase activitybut these preparations were found to degradesodium chondroitin sulfate and potassiumhyaluronate.

REFERENCESBEVERIDGE, W. I. B. 1934 A study of twelve

strains of Bacillus necrophorus with observa-tions on the oxygen intolerance of the organ-ism. J. Pathol. and Bacteriol. 38, 467-491.

ELSON, L. A., AND W. T. J. MORGAN 1933 Acolorimetric method for the determinationof glucosamine and chondrosamine. Bio-chem. J., 27, 1824-1828.

FOLIN, O., AND V. CIOCALTEAU 1927 On tyrosineand tryptophane determinations in proteins.J. Biol. Chem., 73, 627-650.

GIBBONS, R. J., AND J. B. MAcDONALD 1960Hemin and vitamin K compounds as requiredfactors for the cultivation of certain strainsof Bacteroides melaninogenicus. J. Bacteriol.,80, 164-170.

HERBERT, D. 1955 Methods in enzymology, vol.11, pp. 784-785. Edited by S. P. Colowickand N. 0. Kaplan. Academic Press, Inc.,New York.

HOFFMAN, P., A. LINKER, P. SAMPSON, K. MEYER,AND E. D. KORN 1957 The degradation ofhyaluronate, the chondroitin sulfates andheparin by bacterial enzymes (Flavobac-terium). Biochim. et Biophys. Acta, 25,658-659.

JAQUES, L. B., M. MITFORD, AND A. RICKER 1947The metachromatic activity of heparin. Rev.Can. Biol., 6, 740-754.

KORN, E. D., AND A. N. PAYZA 1956 The deg-radation of heparin by bacterial enzymes.II. Acetone powder extracts. J. Biol. Chem.,223, 859-864.

KORN, E. D. 1957 The degradation of heparinby bacterial enzymes. III. A comparison of thedegradation of heparin, hyaluronic acid, andchondroitin sulfate. J. Biol. Chem., 226, 841844.

MANDEL, B., AND E. RACKER 1953 Inhibition ofTheiler's encephalomyelitis virus (GDVIIstrain) of mice by an intestinal mucopoly-saccharide. I. Biological properties andmechanism of action. J. Exptl. Med., 98,399-415.

MARTINEZ, R. J., J. B. WOLFE, AND H. I. NAKADA1959 Purification and properties of the en-zyme chondroitinase. J. Biol. Chem., 234,2236-2239.

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