Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium heparinum. Analyses of the heparin and heparan sulfate degradation products by 13C NMR spectroscopy.

The purification of two heparitinases and a heparinase, in high yields from Flavobacterium heparinum was achieved by a combination of molecular sieving and cation-exchange chromatography. Heparinase acts upon N-sulfated glucosaminido-L-iduronic acid linkages of heparin. Substitution of N-sulfate by N-acetyl groups renders the heparin molecule resistant to degradation by the enzyme. Heparitinase I acts on N-acetylated or N-sulfated glucosaminido-glucuronic acid linkages of the heparan sulfate. Sulfate groups at the 6-position of the glucosamine moiety of the heparan sulfate chains seem to be impeditive for heparitinase I action. Heparitinase II acts upon heparan sulfate producing disulfated, N-sulfated and N-acetylated-6-sulfated disaccharides, and small amounts of N-acetylated disaccharide. These and other results suggest that heparitinase II acts preferentially upon N,6-sulfated glucosaminido-glucuronic acid linkages. The total degradation of heparan sulfate is only achieved by the combined action of both heparitinases. The 13C NMR spectra of the disaccharides formed from heparan sulfate and a heparin oligosaccharide formed by the action of the heparitinases are in accordance to the proposed mode of action of the enzymes. Comparative studies of the enzymes with the commercially available heparinase and heparitinase are described.

The purification of two heparitinases and a heparinase, in high yields from Flavobacterium heparinum was achieved by a combination of molecular sieving and cation-exchange chromatography. Heparinase acts upon N-sulfated glucosaminido-L-iduronic acid linkages of heparin.
Substitution of N-sulfate by N-acetyl groups renders the heparin molecule resistant to degradation by the enzyme. Heparitinase I acts on Nacetylated or N-sulfated glucosaminido-glucuronic acid linkages of the heparan sulfate. Sulfate groups at the B-position of the glucosamine moiety of the heparan sulfate chains seem to be impeditive for heparitinase I action. Heparitinase II acts upon heparan sulfate producing disulfated, N-sulfated and N-acetylated-6-sulfated disaccharides, and small amounts of N-acetylated disaccharide.
These and other results suggest that heparitinase II acts preferentially upon N,6-sulfated glucosaminido-glucuronic acid linkages. The total degradation of heparan sulfate is only achieved by the combined action of both heparitinases. The %! NMR spectra of the disaccharides formed from heparan sulfate and a heparin oligosaccharide formed by the action of the heparitinases are in accordance to the proposed mode of action of the enzymes.
Comparative studies of the enzymes with the commercially available heparinase and heparitinase are described.
Flavobacterium heparinum produces a number of constitutional and induced enzymes which are able to degrade glycosaminoglycans to their basic disaccharide units. Among the constitutional enzymes a chondroitinase AC (1) and a chondroitinase C (2) able to degrade chondroitin sulfates have been identified.
When the bacteria are grown in the presence of chondroitin sulfate or dermatan sulfate two new chondroitinases, namely, chondroitinase B and chondroitinase ABC are induced in the bacteria (3,4). Also, a heparinase and two heparitinases are induced when the bacteria are exposed to heparin, heparan sulfate or their di-and oligosaccharides (5,6).
The presence of these different activities in the bacteria, which act upon closely related substrates, namely heparin and heparan sulfate, poses difficult problems regarding not only their purification but also in obtaining precise information on their mode of action.
Whereas the substrate specificity of the heparinase is reasonably well established (6-13) a clear definition of the specificity of the heparitinases as well as their purification from each other and from the heparinase and the constitutional enzymes is still not completely settled.
The purpose of this paper is the description of a new method for the purification of the enzymes as well as an attempt to define their specificies using natural and modified heparins and heparan sulfates.   to Fransson et al. (22). The quantitation of iduronic and glucuronic acid in the different glycosaminoglycans was performed essentially as described by Kosakai and Yosizawa (23).

RESULTS
Purification of the Enzymes-The fractionation of the 100,000 X g supernatant of F. heparinum extracts by DEAEcellulose column chromatography is shown in Fig. 1. This step does not fractionate the enzymes acting upon heparin and heparan sulfate but removes most of the contaminant proteins together with possible proteolytic enzyme(s) which render the glycosaminoglycanases completely stable for at least 1 year at 5 "C. The fractions containing the enzymatic activities were pooled, concentrated, and fractionated on Bio-Gel A-O.5 m. The individual fractions were assayed with heparin and heparan sulfate (Fig. 2). Three enzymatic activities acting upon heparin and heparan sulfate are completely resolved from each other. The activity eluted between fractions 130-140 acts only upon heparan sulfate. This enzyme was earlier defined as heparitinase I (7). The activity present in fractions 113-122 acts only upon heparin from lung and intestinal mucosa and was defined as the heparinase (6). Finally, the activity present in fractions 90-100 acts upon heparan sulfate and partially upon heparin from bovine intestinal mucosa. Fig. 2 shows that when heparin from bovine lung is used for the assay no significant degradation of this substrate is observed. This enzyme was previously described as heparitinase II (7). These fractions were also tested with other substrates (Fig.  2). The trisulfated disaccharide (AU,2S-GlcNS,GS)' is degraded to disulfated disaccharide (AU-GlcNS,GS) by the fractions containing the heparitinase II which indicates the presence of sulfoiduronate sulfatase previously named disaccharide sulfoesterase (6). Also the disulfated disaccharide (AU-GlcNS,GS) is degraded by GlcNS,GS by the fractions containing the heparinase indicating the presence of a glycuronidase. In order to separate these enzymes, the fractions 90-100 ( Fig.  2) were combined and fractionated on carboxymethylcellulose. The disaccharide sulfoesterase is eluted at 0.03 M, and the heparitinase II is eluted at 0.05 M concentration of ethylenediamine-acetate buffer (Fig. 3). Likewise, the heparinase is eluted together with the glycuronidase, and the heparitinase I overlaps with the chondroitinase AC in the Bio-Gel column (Fig. 2). The fractions containing these enzymatic activities were pooled, applied to a carboxymethylcellulose column, and eluted with increasing concentrations of ethylenediamineacetate buffer. The heparinase is eluted at 0.05 M whereas the glycuronidase is not retained by the column. The heparitinase I is eluted at 0.03 M and the chondroitinase AC between 0.05 and 0.07 M ethylenediamine-acetate buffer (Fig. 3). Table I shows the recovery and degree of purification of the enzymes by these combined procedures. Assuming no loss of activity by the DEAE and Bio-Gel fractionation the heparinase, heparitinase II, and heparitinase I were purified 378-, 950-, and 1855-fold, respectively, by these combined procedures.
The purified enzymes were incubated with all the sulfated disaccharides listed in Table III   disaccharides were desulfated or hydrolyzed by the enzymes indicating that they were essentially free from sulfatases and glycuronidases (results not shown). Chemical Data of the Substrates-The chemical analyses of the substrates used for the studies of the specificities of the enzymes are shown in Table II. The heparin from lung tissue contains BO-90% of the uranic acid residues as iduronic acid and the intestinal mucosal heparin between 65-75% of this uranic acid (24). The estimation of the iduronic acid content of the two heparins (chemical analyses and nuclear magnetic resonance) gave similar results. Conversely, the heparan sulfates used in these experiments contain at least 94% of glucuronic acid, measured by those methods (25).
Substrate Specificity of Heparinase-Bovine lung and intestinal heparins are extensively degraded by heparinase (90 and 60% yields, respectively) as shown in Fig. 4 and Table III. Since heparin contains 65-80% of iduronic acid residues and is extensively degraded it is reasonable to assume that heparinase acts upon glucosaminido-iduronic acid linkages. Conversely, pancreas and lung heparan sulfate, which differs from heparin by the almost exclusive presence of glucuronic acid residues in the molecule (Table II), is a very poor substrate for the enzyme (Fig. 4). The N-sulfate residues in the heparin molecule seem to be essential for the heparinase action since substitution of this sulfate by acetyl groups renders the heparin molecule resistant to the enzyme (Fig. 4). The C-6 sulfate substitution of the hexosamine moiety is not an impediment for the enzyme action since AU,2S-GlcNS is formed from heparin by the heparinase (Table III). Regarding the C-2 sulfate substitution of the uranic acid moiety, no definite information could be obtained with the substrates used. According to other authors (12, 13) the C-2 substitution of the uranic acid moiety of heparin is essential for the heparinase action. The tetrasaccharides also released from heparin by action of heparinase in high yields (Fig. 5 (3), and heparinase (4) in the conditions described in Fig. 4. The products were analyzed as described in Fig. 5  does not act upon non-sulfated uranic acid-glucosaminido linkages. Nevertheless, recent analysis of the tetrasaccharides has shown that the uranic acid is indeed glucuronic acid as previously suggested (6).
A monosaccharide, GlcNS,GS, is also formed from the heparins by action of heparinase in about 3-6% yield. Since no saturated disaccharides were detected as degradation products and the enzyme is free of sulfatases and glycuronidase, it is reasonable to suppose that this monosaccharide is released from the non-reducing end of the molecules by the action of the enzyme. tensively unmodified and N-acetylated heparan sulfate with formation of UV absorbing products. No activity could be detected upon heparin or N-acetylated heparin (Fig. 4). The type of disaccharide products formed by action of the enzyme upon the two heparan sulfates is shown in Fig. 6 and Table  III. AU-GlcNS and AU-GlcNAc are the main products formed by action of this enzyme upon heparan sulfate. A decrease of AU-GlcNS with a correspondingly increase of AU-GlcNAc is observed when N-acetylated heparan sulfate is incubated with heparitinase I (Fig. 6). Note that no or negligible amounts of AU-GlcNAc,GS and AU-GlcNS,GS are formed by action of heparitinase I upon heparan sulfate or N-acetylated heparan sulfate. Since the heparan sulfate used contains more than 90% of its uranic acid residues as glucuronic acid and that the N-acetylated heparin (which contains mostly iduronic acid) is not a substrate for the enzyme it is suggestive that heparitinase I acts upon glucosaminido-glucuronic acid linkages. The findings that no or negligible amounts of AU-GlcNS,GS and AU-GlcNAc,GS are formed by action of heparitinase I from both natural and modified heparan sulfates suggest that the sulfate at the C-6 position of the hexosamine moiety is impeditive for the enzyme action. Specificity of Heparitinase II-Except for lung heparin all the other substrates used in this study were degraded by heparitinse II (Fig. 4). Nevertheless, the extent of degradation and the yield of the disaccharide products formed from the different substrates were quite variable. Thus, intestinal heparin yields 23% of disaccharides.
The remaining oligosaccharide was only degraded by heparinase yielding the characteristic products (Fig. 5, Table III). Heparitinase II acts upon heparan sulfate producing the four main types of disaccharides present in these molecules. Nevertheless, the yield of AU-GlcNac is much lower than that obtained by action of The experiments were performed as described in Figs. 5 and 6 except that the commercial heparinase (0.1 unit) was incubated with lung heparin (upper panel) and commercial heparitinase with pancreas heparan sulfate (lower panel) in 3 mM calcium acetate, pH 7.0, for different periods of time as indicated. St. I, ATetra; St. 2, 1U,E-GlcNS,@; St., mixture of AU-GlcNS,GS and GlcNS.
heparitinase I (Table III). Addition of excess of heparitinase II or longer incubation periods did not increase the yield of this disaccharide.
Only the combination of the two heparitinases degrades completely the heparan sulfate (Figs. 4 and 6). The heparitinase II also acts upon the tetrasaccharides formed from heparin by heparinase to the corresponding disaccharides (6). These combined data suggest that heparitinase II has a broad specificity acting preferentially upon C-6 sulfated glucosaminido-glucuronic acid linkages. Besides the disaccharides, the heparitinase II produces from heparan sulfates the monosaccharide GlcNS,GS (Table III) in low yields. This monosaccharide could be located at the nonreducing end of the molecules since no saturated disaccharides were formed by the action of either heparitinase I or heparitinase II upon the heparan sulfates.
The "'C spectra of the four disaccharides obtained from heparan sulfate by action of heparitinase I and heparitinse II, compared with the profile of the main disaccharide of heparin (AU,2SGlcNS,GS) already reported (26) are shown in Fig. 7. The signal of the C4 of the uranic acid (U4) of all the disaccharides is located at 6107 and corresponds in chemical shift to the unsaturation between C4-C5 of the uranic acid residue. The signals at 623 of AU-GlcNAc and AU-GlcNAc,GS indicate the presence of acetyl residues in these two disaccharides. When the NH, group of the glucosamine is sulfated as in the case of the disaccharide from heparin and the disaccharides AU-GlcNS,GS and AU-GlcNS from heparan sulfate shows the signal of Hl at 692. In the case of the N-acetylated disaccharides this signal is split in two (a91 and 95) corresponding to the o( and /3 configuration of the anomeric carbon of the hexosamine.
This suggests that the sulfate at the C-2 position of the glucosamine maintains this sugar in o( configuration. An important distinguishing feature between the disaccharide obtained from heparin and all the disaccharides obtained from heparan sulfate are the signals of U2, Ul, and U3. The Ul and U3 signals are displaced to low field whereas the signal of U2 is displaced to high field in the heparan sulfate disaccharides due to the absence of the sulfate residue in the uranic acid moiety. Also the displacement to low field of the signal of H6 of AU-GlcNS and AU-GlcNAc shows that these disaccharides are not sulfated at the C-6 of the hexosamine moiety. The finding that the H6S signal of AU-GlcNAc,GS at 667 has the same chemical shift of the signals of the other 6-sulfated disaccharides confirms the previous sugestion (7) that this disaccharide has the sulfate at the C-6 position of the hexosamine moiety. For other details of these analyses see Ref. 28.
The "'C spectra of the anomeric carbons of bovine intestinal heparin and its oligosaccharide produced by action of heparitinase II is shown in Fig. 8. The signal at 103 ppm which corresponds to the C-l of the glucuronic acid is significantly decreased in the oligosaccharide when compared with the intact heparin. This is in agreement with the suggestion that heparitinase II removes preferentially glucuronic acid-containing disaccharides from the heparin molecule. Substrate Specificity of the Commercial Heparinase and Heparitinase-The products formed by action of the commercially available heparinase and heparitinase upon heparin and heparan sulfate are shown in Fig. 9. At short incubation periods, the heparinase produces tetrasaccharides, tri-and disulfated disaccharides from bovine lung heparin. At longer incubation periods there is a decrease of trisulfated disaccharide with a corresponding increase of disulfated disaccharide and glucosamine 2,6-disulfate (GlcNS,GS). This indicates that the heparinase is contaminated with the disaccharide sulfoesterase (an enzyme that removes a sulfate from the C-2 position of the uranic acid moiety) and with glycuronidase (27). The commercially available heparitinase produces upon heparan sulfate-disulfated disaccharide, N-sulfated disaccharide, and glucosamine 2,6-sulfate. It resembles the heparitinase II described in this paper but with low activity and contaminated with glycuronidase. Also both enzymes show a weak chondroitinase activity (results not shown).

DISCUSSION
Functionally purified heparinase, heparitinase I, and heparitinase II free of sulfatases, glycuronidases, and chondroitin sulfate lyases were prepared in high yields by the combination of molecular sieving and ion-exchange chromatography. This methodology also allows the preparation of disaccharide sulfoesterase, glycuronidase, and chondroitinase AC free of the other mucopolysaccharidases.
Higher yields of enzymes than the ones previously described (6) could be obtained by the present method.
The mode of action of the heparinase upon the different glycosaminoglycans is still compatible with the earlier propositions that the enzyme is an eliminase acting upon glucosaminido (Y 1,4-L-iduronic acid linkages of the heparin molecule (6,8,9,12). Thus, bovine lung heparin which contains a large proportion of iduronic acid is more extensively degraded than the intestinal one which contains relatively higher amounts of glucuronic acid residues. The present results also show that replacement of the sulfate radicals from the amino groups of the hexosamines by acetyl groups renders the heparin mole-cule completely resistant to degradation by the heparinase. No evidence for the presence of disaccharides containing sulfate at the C-3 position of the hexosamine, that could theoretically be formed by the action of heparinase or heparitinase II, were obtained. These disaccharides represent less than 0.3% of the heparin molecule (29) and could not be detected by the present methodology.
The lung and pancreas heparan sulfates used in the present study are poor substrates for the heparinase. The small amounts of UV absorbing materials after heparinase upon the heparan sulfate prepared from pancreas could be an indication that a few iduronic acid residues are present in this heparan which is mostly constituted of glucuronic acid residues as revealed by chemical analyses and NMR studies. Both heparan sulfates are extensively degraded by each one of the heparitinases.
N-Acetylated and N-sulfated disaccharides are the exclusive products of the heparitinase I whereas disulfated, N-sulfated, and N-acetylated 6-sulfated disaccharides are the major products of heparitinase II. Small amounts of N-acetylated disaccharide are also formed from the heparan sulfate by heparitinase II. This suggests that this enzyme is somewhat nonspecific having a lower affinity for the Nacetylated regions. The total degradation of the heparan sulfate is achieved only by the combined action of the two heparitinases (Fig. 5, Table III). The heparitinase II also acts extensively upon the intestinal heparin releasing considerable amounts of tri-and disulfated disaccharides and an oligosaccharide which accounts for 75% of the original heparin molecule. This oligosaccharide is in turn degraded by heparinase producing Atetra and trisulfated disaccharide. It thus seems that heparin contains two distinct regions, one of them composed of mostly iduronic acid-containing disaccharides and another composed of glucuronic acid-containing disaccharides. This was also observed by 13C NMR spectra of the oligosaccharide obtained from heparin after heparitinase II degradation which shows a significant decrease of the anomerit carbons of glucuronic acid. This glucuronic acid region seems to be more extense in the intestinal heparin than in the lung heparin as judged by the amount of products formed from the two heparins by action of heparitinase II (Table III) and NMR spectroscopy where glucuronic acid is a minor component of this type of heparin (24).
These combined results suggest that heparitinase II acts preferentially upon N-6-sulfated and/or N-acetylated, 6-sulfated glucosaminido-a-l-4-glucuronic acid linkages. If this is the case the trisulfated disaccharide obtained from heparin by action of this enzyme was derived from the heparin region containing sulfated glucuronic acid residues. This glucuronic acid-containing trisulfated disaccharide has been recently reported by Fedarco and Conrad (30) in a peculiar intracellular heparan sulfate from nuclei of hepatocytes.
In favor of this hypothesis are the findings that the sulfated tetrasaccharides (which contains an internal glucuronic acid residue) formed from heparin by the action of heparinase are excellent substrates for the heparitinase II (6). An alternative possibility would be that heparitinase II is again somewhat nonspecific acting preferentially upon the less sulfated regions of the intestinal heparin. Its lack of activity upon the lung heparin and the heparinase-sensitive region of the intestinal heparin would be related to the special conformation of these more sulfated regions. It is clear that more model compounds and a better understanding of the structure of the substrates are needed to define the specificity of heparitinase II. Except for a few details, the specificity of heparinase and heparitinase II described in this paper agrees with the specificities reported by other authors (8-13). Also, except for the contamination with glycuronidase, sulfatase, and chondroitin sulfate lyase, the commercialy available heparinase and heparitinase, correspond in our hands, to the heparinase and heparitinase II described in this paper. The other commercially available enzymes, namely, heparinase II and heparinase III were not tested with the present methodology.
Nevertheless, as judged by the data presented by other authors (11, 13) these two enzymes seem to differ from the heparitinases I and II described in this paper. Finally, the present results confirm the previous studies that commercial bovine lung heparin has a quite homogeneous structure where more than 90% of its structure is composed of octasaccharide-repeating units (6). This homogeneity has also been stressed by Gatti et al. (24) through 'H and 13C NMR studies of this type of heparin.