Enzymic degradation of ω-heparin (Whale heparin)

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BIOCHIMICA ET BIOPHYSICA ACTA

BBA 25804 ENZYMIC D E G R A D A T I O N OF o~-HEPARIN (WHALE H E P A R I N ) ZENSAKU YOSIZAWA Department of Biochemistry, Tohoku University School of Medicine, Sendai (Japan)

(Received December 2oth, 1966) (Revised manuscript received March 7th, i967)

SUMMARY o~-Heparin isolated from whale organs was digested with crude heparinase from heparin-adapted flavobacteria and also with an eliminase separated from the crude enzyme. o~-Heparin was degraded, b y the crude heparinase, to the three major monosaccharides, an ~-keto acid, N-sulfated glucosamine, and N-,O-disulfated glucosamine. As minor products, N-acetylglucosamine and an N-acetylglucosamine-containing unsaturated oligosaccharide were also found in the digest. The eliminase digestion produced several unsaturated oligosaccharides. Two were indicated to contain unsaturated uronic acid and N-acetylglucosamine with and without O-sulfate. These products were similar to those found in the degradation products of ~-heparin and heparitin sulfate with these enzymes. In addition, a study of the temperature optimum and the effects of inhibitors on the degradation of o-heparin with the eliminase showed o~-heparin to be intermediary between ~-heparin and heparitin sulfate. Moreover, the unsaturated oligosaccharides produced and the changes in the reducing values after the action of these enzymes indicated that o~-heparin was not a mixture of a-heparin and heparitin sulfate, but a complex polysaccharide consistingof an N-acetylglucosamine-containing polysaccharide to which ~-heparin-type polys accharides are attached.

INTRODUCTION ~o-Heparin isolated from whale organs has been shown to be substantially different to the a-heparin of terrestial mammalia 1-3. The former contains N-acetylglucosamine, approx, one-fourth of the total glucosamine, as an integralcomponent of the compound 2,3. I t resembles heparitin sulfate in chemical nature, but differs from it in its biological activitiesL4, molecular weight3,5, 6, and electrophoretic mobilities ~. LINKER and co-workersT,6 reported that crude heparinase from flavobacteria degraded a-heparin and heparitin sulfate to the same major compounds, an a-keto acid, N-sulfated glucosamine and N, 0-disulfated glucosamine, apart from the Nacetylglucosamine of the latter. Furthermore, an eliminase separated from the crude heparinase produced several major compounds containing unsaturated glucuronic acid from these substrates ~. Since o~-heparin has been indicated to be a compound intermediary between Biochim. Biophys. ,4cta, 141 (1967) 600-604

ENZ~'MIC DEGRADATION OF ¢0-HEPARIN

601

a-heparin and heparitin sulfate in its chemical properties 2,~, it was of interest to study the enzymic degradation products. This paper reports the results obtained from a study of the action of these enzymes on ~o-heparin. EXPERIMENTAL

Materials and methods o~-Heparin was kindly supplied b y Dr. T. SHIBATA. a-Heparin, heparitin sulfate, crude heparinase and an eliminase from the heparin-adapted flavobacteria were the materials described previously ~. The methods were as reported beforeS: reducing sugar (as glucose) was determined b y a ferricyanide methodg; paper chromatography was carried out in n-butanolacetic acid-water (50:15 : 35, v/v/v) and/or n-propanol-ethyl acetate-water (7 : i : 2, vfv/v), employing staining reagents of alkaline AgNO31° and also Ehflich's reagent 1~ with or without acetylacetone; incubations were carried out, unless otherwise stated, with io mg of substrate in I ml of o.I M acetate buffer (pH 7.0) with 2 mg of the crude heparinase or i mg of the eliminase at 25°; unsaturated uronic acid was determined by the absorbance at 235 m/~ of aliquots of the incubation mixture diluted with 0.03 M HC1. RESULTS

Action of crude enzyme from heparin-adapted bacteria As reported for ~-heparin and heparitin sulfateT,s, o~-heparin was also degraded by the crude heparinase into three major monosaccharides, which were found to be identical with those obtained from x-heparin and heparitin sulfate, by paper chromatography (Fig. I), and also two minor compounds corresponding to those obtained from heparitin sulfate. No significant increase in absorbance at 235 m/~ was observed. The compounds were isolated by elution from a chromatogram and identified. Compound I reacted with the silver reagent, and a-keto acid reagents such as ophenylendiamine 1~ and semicarbazide la. Compound I I reacted with thesilverreagent, gave 2,5-anhydromannose by deamination with nitrous acid 14, but did not react with the ninhydrin and Ehrlich's reagent without prior hydrolysis. After treatment with o.04 M HC1 at ioo ° for 2 h (ref. I5), it yielded glucosamine. Compound I I I showed the same color reaction as Compound II, however, it gave a spot corresponding to the mono-0-sulfated glucosamine obtained from ~-heparin after mild acid hydrolysis. These findings indicate that ~o-heparin was also degraded to an a-keto acid (Compound I), N-sulfated glucosamine (Compound II) and N,0-disulfated glucosamine (Compound III). Besides these compounds, N-acetylglucosamine (Spot A) and an oligosaccharide (Spot B) containing N-acetylglucosamine and unsaturated uronic acid were also detected as minor products. These two compounds were also present, at higher concentrations, in digests of heparitin sttlfate (Fig. I). The increase in the amount of reducing sugar released with time is shown in Fig. 2. It can be seen from Fig. 2, that o-heparin was degraded more slowly than a-heparin during approx. I0 h incubation; however, the amount of reducing sugar Biochim. Biophys. Acta, 141 (1967) 6oo-6o 4

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Fig. 3- D i g e s t i o n of ~ - h e p a r i n , a-heparin, a n d h e p a r i t i n sulfate w i t h t h e eliminase. - . . . . . . , C h a n g e in u l t r a v i o l e t a b s o r p t i o n ; - - , c h a n g e in r e d u c i n g sugar. A, ~o-heparin; O, ~¢-heparin; × , h e p a r i t i n sulfate. Fig. 4. T r a c i n g of a c h r o m a t o g r a m s h o w i n g t h e p r o d u c t s o b t a i n e d f r o m ¢o-heparin (w), ~¢-heparin (,¢) a n d h e p a r i t i n sulfate (HS) b y t h e eliminase digestion for z 5 h. H a t c h i n g indicates u l t r a v i o l e t a b s o r p t i o n b y t h e s p o t s otherwise detected b y silver reagent. Spots a a n d b also r e a c t e d w i t h E h r l i c h ' s r e a g e n t w i t h or w i t h o u t acetylacetone. Solvent, n - b u t a n o l - a c e t i c a c i d - w a t e r ( 5 o : i 5 : 3 5 ,

v/v/v). Biochim. Biophys. Acta, 141 (1967) 6oo-6o 4

ENZYMIC

DEGRADATION

OF (O-HEPARIN

603

released from the former was greater than from the latter after a more prolonged incubation time. Digestion with elimir~ase The increase in the amount of reducing sugar released, the change of absorbance at 235 m~ with time, and a paper chromatogram of the digest of o~-heparin after 15 h incubation with the eliminase are shown in Figs. 3 and 4, respectively, together with those of ~-heparin and heparitin sulfate. The data show that the present preparation of eliminase was contaminated with a certain amount of a glucuronidase which hydrolyzed the unsaturated uronides to monosaccharides s. The absorbance at 235 mt~ and the reducing sugar values of o~-heparin were less than those of e-heparin within approx. 12 h, but these values of the former increased beyond those of the latter on further incubation as observed for the crude heparinase. The degradation products of o~-heparin with eliminase gave spots corresponding to those from both e-heparin and heparitin sulfate. Compounds a and b, which were positive for Morgan-Elson reaction and also for the silver reaction, but negative for ninhydrin reaction, did not produce 2,5-anhydromannose after deamination with nitrous acid. Since these compounds showed ultraviolet absorption, they were indicated to contain ~, E-unsaturated uronic acid. The Compound a from heparitin sulfate was shown to be sulfate free s, but the Compound b contained sulfate (A. LINKER AND P. HOVlNGH, unpublished data). Compound b was shown to be the same as the Compound B obtained with crude heparinase. Compounds c, d, e, and f are not yet fully characterized, but are thought to be sulfated, unsaturated oligosaccharides s. Compounds a and b, particularly Cmnpound b, from ~-heparin could not be detected by paper chromatography in the early stages (3 h or 6 h) of the digestion, however, other unsaturated oligosaccharides were readily detected. All of the unsaturated oligosaccharides obtained from ~-heparin and heparitin sulfate (Fig. 4) were readily detected early in the digestion. The temperature optimum for the eliminase digestion was shown to be between 25 ° and 3 °o with ~-heparin and at 37 ° with heparitin sulfate s. It was found to be between 3 °0 and 34 ° with ~-heparin. Hg ~+ was inhibitory to a concentration of IO-5 M with e-heparin as substrate s, and 65 % inhibition was observed at IO-* M with o~-heparin. EDTA showed 9 ° % inhibition at lO-3 M with ~-heparin, but was without effect on the degradation of heparitin sulfate s. This compound showed approx. 6o % inhibition at ~o-a M with ~-heparin. DISCUSSION

Some information on the structure of ~o-heparin can be obtained from the results of the present study. The presence of the same major degradation products, an a-keto acid, N-sulfated glucosamine and N,O-disulfated glucosamine, in digests of ~o-heparin, a-heparin and heparitin sulfate with the crude heparinase points to a certain similarity in structure of these three compounds and the closer similarity of ~o-heparin to heparitin sulfate, because of the other two identical products, N-acetylglucosamine and an N-acetylglucosamine-containing unsaturated oligosaccharide. This similarity is also confirmed b y the finding that the digest of ~o-heparin with eliminase contained the compounds found in the degradation products of a-heparin and heparitin sulfate. Biochim. Biophys. Acla, 141 (~967) 6oo-6o4

604

Z. YOSIZAWA

Furthermore, the temperature optimum and the effects of the inhibitors on the degradation of o~-heparin with the eliminase showed that ~o-heparin is a compound intermediary between ~-heparin and heparitin sulfate. o~-Heparin was degraded more slowly than ~-heparin in the early stage of the incubation with both tile crude heparinase and the eliminase, although the degradation of the former was the more complete after prolonged incubation periods. Heparitin sulfate was degraded most extensively from the early stages of incubation (Figs. 2 and 3)Moreover, the N-acetylglucosamine-containing unsaturated oligosaccharides (Compounds a and b) produced by the eliminase from ~o-heparin could not be detected in the early stage of the incubation, while other unsaturated oligosaccharides were readily detected. All of the unsaturated oligosaccharides found in the digests from ~-heparin and heparitin sulfate could be observed distinctly from an early period in the digestion. These findings suggest that these enzymes mainly degrade the ~-heparin-type core of ~o-heparin in the early period of the incubation, followed by digestion of the N-acetylglucosamine-containing core. This is again indicative that ~o-heparin is not a mixture of ~-heparin and heparitin sulfate, but a complex polysaccharide consisting of an N-acetylglucosamine-containing polysaccharide to which ~-heparin-type polysaccharides are attached. The good yield of the ~, E-unsaturated uronides indicates the presence of I-~4 linkages in ~o-heparin, as the major bond, similar to ~-heparin and heparitin sulfate, as previously describeds. ACKNOWLEDGEMENTS

The author thanks Dr. A. LINKER,Departments of Biochemistry and Pathology, University of Utah College of Medicine, and the Veterans Administration Hospital, Salt Lake City, Utah, U.S.A., for providing the author with the opportunity to work in his laboratory and for his interest throughout this work. The author is also indebted to Dr. T. SHIBATAand Taiyo Fishery Co. Ltd., Japan, for this investigation. This work was supported in part by Grant HE 06o80 from the U.S. Public Health Service, U.S.A. REFERENCES i K. HASHIMOTO, M. MATStINO, Z. YOSIZAWAAND T. SHIBATA, Tohohu J. Exptl. Med., 81 (1963) 93. 2 Z. Y'OSIZAWA,Biochem. Biophys. Res. Commun., 16 (1964) 336. 3 T. KoToKtL Z. YOSIZAWAAND F. YAMAUCHI, Arch. Biochem. Biophys., in the press. 4 M. MATSUNO AND K. HASHIMOTO, Tohoku J. Expa. Med., 83 (1964) 143. 5 D. ~-I. BROWN, Proc. Natl. Acad. Sci. U.S. 43 (1957) 783. 6 J. KNECHT AND A. DORFMAN, Biochem. Biophys. Res. Commun., 21 (1965) 509. 7 A. LINKER AND P. SAMPSON, Biochim. Biophys. Acta, 43 (196o) 366. 8 A. LINKER AND P. HOVlNGVI,J. Biol. Chem., 24o (1965) 3724 . 9 M. M. RAPPORT, K. MEYER AND A. LINKER, J. Biol. Chem., 186 (195 o) 615. xo W. E. TREVELYAN, D. P. PROCTOR AND J. S. HARRISON, Nature, 166 (195 o) 444. i i S. M. PARTRIDGE, Biochem. J., 42 (I948) 238. 12 M. C. LANNING AND S. S. COHEN, J. Biol. Chem., 189 (1951) lO9. 13 H. E. UMBAEGER AND B. MAGASANIK, J. Am. Chem. Soc., 74 (I952) 4253 . 14 Z. YOSIZAWA, Tohoku J. Exptl. Med., 74 (1961) 69. 15 I. DANISHEFSKY, H. B. EIBER AND J. J. CARE, Arch. Biochem. Biophys., 9 ° (I96o) 114.

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