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Inhibition of human leukocyte elastase activity by heparins: influence of charge density
BB
Bioch et Biophysica AEta ELSEVIER
Biochimica et Biophysica Acta 1290 (1996) 299-307
Inhibition of human leukocyte elastase activity by heparins: influence of charge density Nicola Volpi * Department of 'Biologia Animale', Chair of Biological Chemistry, Uni~,ersityof Modena, 1-41100Modena, Italy Received 29 February 1996; accepted 26 March 1996
Abstract
Heparins with different structures and physico-chemical properties were evaluated for their capacity to inhibit human leukocyte elastase activity in vitro by using a chromogenic substrate. Heparin from bovine intestinal mucosa and heparan sulfate from bovine spleen were extracted and purified, and their purity, structures, and physico-chemical properties were evaluated. Slow moving and fast moving heparin species were obtained by selective precipitation as barium salt, and partially desulfated and re-N-sulfated heparin was produced by chemical modifications. Heparins with different molecular mass (from 950 to 7820), narrow polydispersity and the same charge density were produced by a chemical depolymerization process in the presence of free radicals, and further gel-permeation chromatography. Heparins strongly inhibit elastase activity, and there is a significant linear dependence between charge density (sulfate-to-carboxyl ratio) and enzymatic activity. We also found a significant linear correlation between the percentage of N-sulfate groups and increased inhibition of elastase activity and between the percentage of iduronic acid and enzymatic activity. Heparin samples with a Mr greater than about 2000-3000 inhibit the HLE activity to the same extent (about 59%) whilst two fractions with a Mr of 1530 (29% inhibition of HLE activity) and 950 (4% inhibition of HLE activity) have less capacity to produce a decrease in the enzymatic activity. Keywords: Elastase; Glycosaminoglycan; Heparin; Heparan sulfate; (Human leukocyte)
1. Introduction
Human leukocyte elastase [EC 3.4.21.37] (HLE), a cationic glycoprotein with p I near 10 and 30 kDa formed by 218 amino-acid residues with 19 arginines [1], is an essential component of polymorphonuclear leukocytes. This serine proteinase has several physiologic functions including proteolysis of phagocytosed proteins and plasma
Abbreviations: HPSEC, high-performance size-exclusion chromatography; Mr, relative molecular mass; SAX-HPLC, strong anion-exchange high-performance liquid chromatography; LMM, low molecular mass; HLE, human leukocyte elastase; GAGs, glycosaminoglycans; SLAPN, N-succinyl-(L-alanine)3-p-nitroanilide; ADiH-0S, 2-acetarnido-2-deoxy-4O-(4-deoxy-ot-L-threo-hex-4-enepyranosyluronic acid)-o-glucose; ADiHNS, 2-deoxy-2-sulfamino-4-O-(4-deoxy-et-L-threo-hex-4-enepyranosyluronic acid)-o-glucose; ADiH-6S, 2-acetamido-2-deoxy-4-O-(4-deoxyet-L-threo-hex-4-enepyrarmsyluronic acid)-6-O-sulfo-D-glucose; ADiH2,NdiS, 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-ct-L-threo-hex-4enepyranosyluronic acid)~D-glucose; ADiH-N,6diS, 2-deoxy-2-sulfamino4-O-(4-deoxy-et-L-threo-hex-4-enepyranosyluronic acid)-6-O-sulfo-D-glucose; ADiH-triS, 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-ot-Lthreo-hex-4-enepyranosyluronic acid)-6-O-sulfo-D-glucose. * Corresponding author. Fax: + 39 59 581020.
proteins (antithrombin III, immunoglobulins and components of the complement system), degradation of connective macromolecular components such as elastin, interstitial and type IV collagens, proteoglycans, laminin, and fibronectin [2,3]. Acute tissue inflammation is associated with the migration and activation of polymorphonuclear neutrophils followed by the release of HLE (present in millimolar amounts in the azurophil granules), which is able to cause tissue damage [3]. An efficient anti-HLE control system is present at inflammatory sites to prevent undesirable degradation of proteins. HLE inhibitors include otl-proteinase inhibitor, ot2-macroglobulin and mucus proteinase inhibitor. The inefficient or defective control of HLE by naturally occurring inhibitors may lead to diseases such as emphysema, rheumatoid arthritis and periodontitis [4]. Due to its strongly cationic nature, human leukocyte elastase binds to and its activity is inhibited by a variety of polyanions, including bacterial polyanions [5], synthetic RNA homopolymers [6], mucus glycoprotein (mucin) [7], and sulfated glycosaminoglycans (GAGs) [8]. Sulfated GAGs, heparin and heparan sulfate, keratan sulfate, and
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N. Volpi / Biochimica et Biophysica Acta 1290 (1996) 299-307
chondroitin sulfates, are complex heteropolysaccharides covalently bound to proteins forming proteoglycans localized at extracellular and cellular levels. They interact with several proteins (for reviews see [9-11]), such as proteinase inhibitors, plasma lipoproteins, lipolytic enzymes. growth factors, extracellular matrix proteins, and others. Interaction with proteins and control of their activities determine several physiological (and pharmacological) activities of GAGs, such as anticoagulant, antithrombotic and antiatherosclerotic properties. Purified preparations of heparin (and derivatives such as low molecular mass (LMM)-heparins and oligosaccharides), heparan sulfate, chondroitin sulfate A and C, and dermatan sulfate (chondroitin sulfate B) prepared by extraction and purification from animal tissues, inhibit HLE activity to different extents depending on the structure and physico-chemical properties of GAGs [12,13]. Furthermore, LMM-heparin influences the kinetics of HLE inhibition via a mucus proteinase inhibitor [14]. The use of natural and synthetic (such as N-oleoyl derivatives of heparin) HLE inhibitors has been proposed as a strategic therapy to prevent protein degradation during the healing phase of emphysema or rheumatoid arthritis [15]. Heparin interacts with HLE by a tight binding, hyperbolic, noncompetitive inhibition mechanism [12,15], and a higher degree of sulfation is expected to be accompanied by lower K i values. On the other hand, M r is another key factor that can influence the interaction between heparin and HLE [ 12,13]. We studied the effect of heparin, heparan sulfate and derivatives with different molecular mass (Mr), charge density and chemical properties on HLE activity in an effort to clarify possible structure-function relationships.
2. Materials and m e t h o d s
2.1. Materials Human leukocyte elastase [E.C. 3.4.21.37] (370 units/mg of protein) was obtained from Sigma. Chromogenic substrate N-succinyl-(L-alanine)3-p-nitroanilide (SLAPN) was from Calbiochem. Papain [E.C. 3.4.22.2], trypsin [E.C. 3.4.21.4], collagenase [E.C. 3.4.24.3], DNAse I [E.C. 3.1.21.1] and RNAse A [E.C. 3.1.27.5] were purchased from Sigma. Hyaluronic acid from human umbilical cord was from Sigma. Heparinase I from Flavobacterium heparinum [E.C. 4.2.2.7.] (2000 units/mg of protein; one unit forms 1.0 nanomol of unsaturated uronic acid per min at pH 7.0 at 37°C), Heparinase II from Flavobacterium heparinum [no assigned E.C. number] (47 units/mg of protein; one unit forms 0.1 Ixmol of unsaturated uronic acid per hour at pH 7.0 at 25°C), Heparinase III from Flavobacterium heparinum [E.C. 4.2.2.8] (215 units/mg of protein; one unit forms 0.1 Ixmoi of unsaturated uronic acid per hour at pH 7.0 at 25°C) were
obtained from Sigma. Unsaturated heparin/heparan sulfate disaccharides (ADiH-0S, ADiH-NS, ADiH-6S, ADiH2,NdiS, ADiH-N,6diS and ADiH-triS, see abbreviations) were obtained from Seikagaku (Tokyo, Japan). Diatomite filters (High Performance Filter Aids) were from Dicalite (Los Angeles, CA, USA). Ecteola-cellulose was from Serva (Heidelberg, Germany). Cation-exchange resin Amberlite IR-120 in Na + form and anion-exchange resin Amberlite IRA-400 in OH- form were from Supelco (Bellefonte, USA). Chelex 100 chelating resin was from Bio-Rad (Richmond, CA, USA). Bio-Gel P6 (particle size range, 45-90 txm; molecular mass range from 1000 to 6000) and Bio-Gel P2 (particle size range, 45-90 I~m; molecular mass range from 100 to 1800) were from BioRad (Richmond, CA, USA). Column Protein Pak 125 (300 × 7.8 ram; particle size: 10 txm; molecular mass ranges: native globular from 2000 to 80000 and random coil from 1000 to 30000; cod. 84601) and Protein Pak 300 (300 × 7.5 mm; particle size: 10 p~m; molecular mass ranges: native globular from 10 000 to 400 000 and random coil from 2000 to 150000; cod. T72711) were from Waters (Milford, USA). 10 I~m Spherisorb SAX (trimethylammoniopropyl groups Si-CH2-CH2-CH2-N+(CH3)3 in CI form) was from Phase Separations (Deeside Industrial Park, Deeside Clwyd, UK). Multiphor II electrophoretic cell (from Pharmacia LKB Biotechnology, Uppsala, Sweden) was used for agarose-gel electrophoresis. Polyacrylamide-gel electrophoresis was performed on a Bio-Rad (Richmond, CA, USA) Protean Ilxi vertical-slab-gel unit connected to a Pharmacia-LKB (Pharmacia, Uppsala, Sweden) 2219 Multitemp II thermostatic circulator. Power was supplied by an LKB 2197 power supply. High purity agarose, acrylamide, bisacrylamide, and barium acetate were from Bio-Rad. 1,2-Diaminopropane and cetyltrimethylammonium bromide were from Merck (Darmstadt, Germany). Bromophenol blue and Azure A were from Sigma (St. Louis, MO, USA). The densitometric instrument was composed of a Macintosh Ilsi computer interfaced with Microtek Color Scanner from Microtek (Hsinchu, Taiwan). IMAGEprocessing and analysis program, Ver. 1.41 from the Jet Propulsion Laboratory of NASA (Florida, USA) was used for densitometric analysis of agarose-gel electrophoretic bands. All the other reagents were analytical grade.
2.2. Extraction and purification of heparin Bovine intestinal mucosa was ground and treated with papain at 65°C for 12 hours in a reaction vessel. After heating to 100°C for 30 rain, the mixture was brought to pH 9.0 by adding 2 N NaOH. After 24 h at 40°C, the product (brought to pH 6.0 with 2 N acetic acid) was filtered on a diatomite filter, and the solution containing polysaccharides was percolated through the ecteola-cellulose resin column. Peptides, nucleic acids and oligosaccharides were eluted with 0.2 M NaC1. Heparin was
N. Volpi/ Biochimicaet BiophysicaActa 1290(1996)299-307 eluted with 3.0 M NaCI, and the recovered solution was added to 1.5 vols. of acetone. The precipitate was solubilized in water and any dermatan sulfate present was removed by selective precipitation as its copper salt [16]. Heparin was further purified by sequential precipitation in 0.6 vol. of acetone [17] and transformed into its sodium salt on Amberlite IR-120 cation-exchange resin in Na + form. Crude heparin sodium salt was collected by precipitation with 2.0 vols. of acetone and dried. This preparation of heparin was called Hep I. A sample of purified heparin from bovine intestinal mucosa was a gift of OmniaBios (Brescia, Italy) and called Hep II.
2.3. Purification of fast moving and slow moving heparin species The slow moving and fast moving components of heparin were purified as their barium salts at different temperatures, as previously reported [18,19]. Purified bovine intestinal mucosa heparin was dissolved in water, and barium acetate (5%) was added slowly with stirring (the pH of the solution was adjusted to 6.0-7.0). After heating to 50-70°C, the solution was left at room temperature (2025°C) for 24 h. The precipitate obtained was solubilized in water and transformed into its sodium salt on Amberlite 1R-120 resin. The crude slow moving heparin species sodium salt was collected by precipitation with 2.0 volumes of acetone and dried. The supernatant was maintained at 5°C for 24 h and the precipitate was collected by centrifugation at 5°C. The fast moving species barium salt was purified as reported for slow moving species.
2.4. Extraction and purification of heparan sulfate B o v i n e s p l e e n was g r o u n d in 500 ml chloroform/methanol 2:1 (v/v). After centrifugation at 5000 rpm for 20 min, the pellet was dried and suspended in double distilled water (500 rnl) and treated with proteolytic enzymes; papain (1 g), trypsin (100 mg) and collagenase (100 mg) were incubated at their optimum temperature (60°C for papain and 37°C for the other proteinases) for 24 h in a reaction vessel. After heating at 100°C for 30 min, DNAse I (100 mg) and RNAse A (10 mg) were added, and the mixture was treated at 37°C for 24 h. After heating to 100°C for 30 min, the mixture was brought to pH 9.0 by adding 2 N NaOH. After 24 h at 40°C, the product (brought to pH 6.0 with 2 N acetic acid) was filtered on a diatomite filter, and 1.5 vol. of acetone was added to precipitate heteropolysaccharides. The recovered precipitate was dried at 60°C for 24 h, dissolved in double distilled water and percolated through the ecteola-cellulose resin column. Peptides, proteins, nucleotides and oligosaccharides still present were eluted with 0.2 M NaCI. Heparan sulfate was eluted with 1.8 M NaC1; 1.5 vol. of acetone were added to the recovered solution and the
301
precipitate was dried at 60°C for 24 h. Heparan sulfate was percolated through the Amberlite IR-120 resin and transformed into heparan sulfate sodium salt.
2.5. Chemical modification of heparin Partial desulfation and re-N-acetylation were essentially performed as reported by Danishefsky et al. [20]. Hep I was both N- and O-desulfated by treating for 30 min with 0.2 N HC1 at 70°C. This derivative was re-N-acetylated by adding acetic anhydride (1 ml per 10 g of polysaccharide) at 25-30°C maintaining the pH in the range of 7 - 8 with NaOH. The reaction takes approx. 30 min under agitation. Modified heparin was recovered by precipitation with 1.5 vol. of acetone, dried and transformed to its sodium salt on Amberlite IR- 120 resin.
2.6. Preparation of LMM-heparin fractions by chemical process in the presence of free radicals Different LMM-fractions were obtained from purified heparin (Hep I) by a controlled chemical depolymerization process induced by free radicals in the presence of copper salt, as previously reported [21,22]. Ten grams of heparin and 0.4 g of copper acetate monohydrate (0.02 M) were dissolved in 100 ml of water in a reaction vessel. Temperature was kept at 60°C and the pH adjusted to 7.5 by adding 1 M NaOH solution. A 9% ( v / v ) hydrogen peroxide solution was added at a rate of 10 ml/h. The reaction for heparin was stopped at different times, and at the end of the reaction, the Chelex 100 chelating resin was utilized to remove pollutant copper from the product, and the strong anion-exchange resin Amberlite IRA-400 in O H - form was used to remove acidic pollutants. The pH of the percolate was adjusted to 6.0 by adding excess acetic acid and then two volumes of acetone were added. The precipitate was collected by filtration, washed with acetone and dissolved again in 100 ml of water. One gram of sodium acetate was added to this solution, and then different sodium salt M r fractions were precipitated by 2.0 vols. of acetone. The precipitate was collected and dried. Heparins with different M r were prepared by stopping the chemical depolymerization process at different times. The different molecular mass fractions were further fractionated on a column packed with Bit-Gel P6 (2 × 80 cm) and eluted with 1 M NaC1. The fractions (determined by HPSEC [23]) were desalted on Bit-Gel P2 (1 × 90 cm) and lyophilized.
2.7. Agarose-gel electrophoresis The purity of Hep I and II, slow moving and fast moving heparin species, heparan sulfate and partial desulfated and re-N-acetylated heparin (PDSNAcHep) were analyzed by agarose-gel electrophoresis in barium acetate/1,2-diaminopropane as reported elsewhere [17,19]. The first run was performed in 0.04 M barium acetate buffer (pH 5.8) for 20 min at 60 mA; the second run was
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in 0.05 M 1,2-diaminopropane buffered at pH 9.0 with acetic acid for 45 min at 50 mA. After migration, the plate was soaked in cetyltrimethylammonium bromide (0.1% solution) for about 3 h, dried and then stained with Toluidine blue (0.2% in ethanol/water/acetic acid, 50:49:1 v / v / v ) for 30 min and destained with ethanol/water/ acetic acid 50:49:1 ( v / v / v ) . The slow moving and fast moving component percentages of Hep I and II were analyzed quantitatively by a densitometer and the amounts evaluated by specific calibration curves.
2.8. Determination of peak
Mr
Heparinase I1 and 0.5 units of Heparinase III in 100 mM (pH 7.0) acetate buffer in the presence of 2.5 mmol calcium acetate. The reactions were stopped, after 24 h at 37°C, by boiling 1 min. Constituent disaccharides were determined by SAXHPLC at 232 nm. Isocratic separation was run for 0 to 5 rain with 0.2 M NaCI (pH 4.00); linear gradient separation for 5 to 90 min with 100% 0.2 M NaCI (pH 4.00) to 100% 1.2 M NaC1 (pH 4.00). Flow was 1.4 ml/min. I separated the nonsulfated and variously sulfated disaccharides according to the standards and retention times by Seikagaku Kogyo (see Table 1 and structural formula). The amount of each identified disaccharide was determined by purified standards and reported as weight percent. Under experimental conditions, the simultaneous cleavage of heparin fractions with heparinase I, II and III produces about 75-80% disaccharides and 20-25% oligosaccharides still resistant to treatment with heparinases. Thus, the percentage of each disaccharide was calculated considering the sum of the masses of disaccharides as 100%.
values
HPLC equipment was manufactured by Jasco, Tokyo, Japan (pump mod. 880 PU; system controller mod. 801 SC; ternary gradient unit mod. 880-02; Rheodyne injector equipped with a 100 ~1 loop; UV detector, mod. 875 UV). The mobile phase was composed of 125 mM Na2SO 4 and 2 mM NaH2PO 4 adjusted to pH 6.0 with 0.1 M NaOH. Flow rate was 0.9 ml/min with a back pressure of 25 k g / c m z. Column Protein Pak 125 and Protein Pak 300 were assembled in series. The peak M r (mean value) was determined by a calibration curve plotted with glycosaminoglycans standards, as reported elsewhere [23].
2.10. Determination of sulfate-to-carboxyl ratio Sulfate and carboxyl groups were determined by potentiometric titration [24] with 0.1 N NaOH in water/dimethylformamide of the glycosaminoglycan acid forms obtained by removing cations on the strong exchanger resin Amberlite IR-120 in H + form. The sulfate-to-carboxyl ratio was also determined by enzymatic degradation after HPLC separation of constituent disaccharides. The ratio was calculated by consid-
2.9. Constituent disaccharide quantitation of heparins, heparan sulfate, ,fast moving and slow moving species and partial desulfated and re-N-acetylated heparin 200 ~g (20 m g / m l in H20) of each species were incubated with 18.5 units of Heparinase I, 0.5 units of
Table 1 Disaccharide composition (expressed as weight percent), physico-chemical properties (M r, charge density, uronate percentages, N-sulfate and N-acetyl groups percentages) and anticoagulant activity of purified heparins (Hep I and Hep II), slow moving heparin component (SMHep), fast moving heparin species (FMHep), beef spleen heparan sulfate (HS) and partially desulfated and re-N-acetylated heparin (PDSNAcHep)
ADiH-0S ADiH-NS ADiH-6S A DiH-2,NdiS A DiH-N,6diS A DiH-2,N,6triS M r (X 1000) SO~/COO- ~ NS% ~ NAc% ~ IdoA% ~ GIcA% a APTT ( I U / m g )
RI
R2
R6
COCH~ SO 3 COCH 3 SO~SO~SO~-
H H H SO 3 H SO~-
H H SO3 H SO~SO~-
Hep I
Hep II
3.8 2.2 2.0 I9.0 I 1.0 62.0 13.10 2.50 94.2 5.8 90.0 10.0 170
SMHep
4.3 1.8 2.4 17.2 13.5 60.8 12.35 2.45 93.3 6.7 88.2 I 1.8 155
2.0 0.9 0.2 15.3 10.3 71.2 14.90 2.66 97.5 2.5 95.6 4.4 > 190
FMHep 8.9 5.5 2.5 22.9 22.4 37.7 7.92 2. l 1 88.6 11.4 81.6 18.4 58
HS 21.0 21.1 24.3 11.2 4.7 5.7 38.30 0.94 54.7 45.3 17.7 82.3 < 0.5
PDSNAcHep 33.1 11.2 6.6 10.5 7.0 31.4 8.70 1.47 60.1 39.9 88.2 11.8 10
For structures of the disaccharides see formula:
0;~
H•O•2 OR
~;H20R6 ~
a S 0 3 / C 0 O - ratios are reported as average values from potentiometric and enzymatic determinations; ldoA%, iduronic acid percentage; GlcA%, glucuronic acid percentage; NS%, N-sulfate group percentage; NAc%, N-acetyl group percentage.
N. Volpi / Biochimica et Biophysica Acta 1290 (1996) 299-307
ering the presence and the percentage of carboxyl and sulfate groups for each disaccharide.
2.11. Determination of iduronate / glucuronate ratio Uronate ratio of heparins, fast moving and slow moving species and heparan sulfate was evaluated by repeated acid hydrolysis and deaminative cleavage. The split uronic acids were then separated and quantified by ion-exchange chromatography [25].
2.12. Determination of N-acetyl group of heparins, heparan sulfate and derivatives The amount of N-acetyl groups were determined as reported elsewhere [26] and by separation and quantitation of constituent disaccharides (see Section 2.9).
2.13. Polyacrylamide-gel electrophoresis The gel was prepared essentially as reported by Rice et al. [27]. The resolving gel and lower buffer chamber contained 0.1 M boric acid/0.1 M Tris/0.01 M disodium EDTA buffer (pH 8.3). The stacking gel was prepared in the same buffer adjusted to pH 6.3 with HCI. The upper buffer chamber contained 0.2 M Tris/1.25 M glycine hydrochloride (pH 8.3). Gels were poured vertically, using a gel-pouring stand, between glass plates (20 X 16 cm) separated by 1.0 mm spacers. Gradient gels were poured by adding 20 ml of 12% total acrylamide (acrylamide + bisacrylamide) solution to the front chamber of the gradient apparatus and 20 ml of 25% total acrylamide solution to the back chamber. A 5 ml portion of stacking-gel solution containing 5% total acrylamide was applied to the top of the resolving gel. Various LMM-heparin samples (5-10 p,l, 10 m g / m l of distilled water) were combined with 20 pA of 50% ( w / v ) sucrose solution containing 0.1% ( w / v ) Bromophenol blue. Electrophoresis was performed at 50 mA (400-500 V) for 2.5-3.0 h at 5°C. Gels were removed from the glass plates and stained for 30 min in 0.08% (w/v) Azure A in water. Destaining was accomplished with rinsing.
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sample (heparin, heparan sulfate or derivatives at different concentration) were mixed with 165 p,1 of 0.05 M Tris-HC1, 0.05 M NaCI (pH 8.0), 15 pA of HLE at a concentration of 1 mU/pA in the same buffer were added, and the solution was incubated for 60 s at 25°C. 20 pA of 5 mM of the chromogenic substrate SLAPN (dissolved in 10% of dimethylsulfoxide in Tris-HC1 buffer) were added and the reaction incubated at 25°C for 24 h. The reaction was stopped by adding 100 pA of 0.5 M acetic acid, and the rate of hydrolysis was monitored at 405 nm versus a blank composed of chromogenic substrate in buffer. The results are expressed as percentage inhibition in comparison with controls (without addition of GAGs). To demonstrate the ionic interactions between enzyme and heparins (Hep I and Hep II at a concentration of 1 p,g), assays were performed with buffer (0.05 M Tris-HC1, pH 8.0) containing increasing ionic strength of NaC1, 0.2 M, 0.4 M, 0.6 M and 0.8 M, and compared with controls composed of 0.05 M Tris-HC1, 0.05 M NaC1, pH 8.0 buffer.
3. Results
3.1. Determination of structures and properties of heparins, heparan sulfate and deriuatives The purity of the various glycosaminoglycans was carefully evaluated by different methods. Electrophoretic separations in agarose-gel (Fig. l) and in Titan III [30] show 'pure' preparations of heparins (Hep I and II), fast moving species, native heparan sulfate and partially desulfated and re-N-acetylated heparin. Heparin is formed by two components [18], a slow and fast moving moiety, and Hep I has about 40% of the first and 60% of the second, whilst Hep II shows about 32% of the most highly sulfated and higher-Mr species and 68% of the less sulfated and lowerM r species.
2.14. Determination of in vitro anticoagulant activity The anticoagulant activity of heparin, fast moving and slow moving species, bovine spleen heparan sulfate, partially desulfated and re-N-acetylated heparin, and different LMM-heparins was determined as Activated Partial Thromboplastin Time activity (APTT activity) [28]. The APTT activity was determined versus the WHO IV International Heparin Standard [29] and expressed as IU/mg.
2.15. HLE chromogenic assay This assay was performed essentially as reported by Walsh et al. [13] with minor modifications. 100 ~1 of
Fig. 1. Agarose-gel electrophoretic separation of glycosaminoglycans. 1 Glycosaminoglycan standards (A - slow moving species, B - fast moving species, C - dermatan sulfate, D - chondroitin sulfate); 2 Hep I, 5 ~.g; 3 Hep II, 5 lug; 4 slow moving heparin, 5 p.g; 5 fast moving heparin, 5 p.g; 6 heparan sulfate, 5 ~,g; and 7 partially desulfated and re-N-acetylated heparin, 5 lug. The arrow indicates the direction of electrophoretic migration.
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N. Volpi / Biochimica et Biophysica Acta 1290 (1996) 299-307
The slow and fast moving species were prepared by fractionation of heparin barium salt at different temperatures [18,19]. The slow moving heparin preparation has about 7% of the fast moving species whilst the fast moving heparin is composed of 100% pure component, as tested by electrophoresis (Fig. 1). Agarose-gel electrophoresis of bovine spleen heparan sulfate shows an electrophoretic band with mobility similar to fast moving heparin (Fig. 1). Analysis of the constituent disaccharides (see below) demonstrates that the fast moving component of heparin and heparan sulfate are different macromolecules in spite of their similar agarose-gel electrophoretic mobility. The possible presence of peptides or proteins in preparations of heparins and heparan sulfate used in the experiments was tested by the Folin phenol reagent [31], confirming the absence (less than 0.1% w / w ) of peptides or proteins. Hep I has an M r = 13100, with 90% of the heteropolysaccharide chains ranging from M r = 23700 to M r = 6000, a sulfate-to-carboxyl ratio of 2.50 and an anticoagulant activity of 170 IUflmg. The simultaneous cleavage of heparin with heparinase I, II and III produces, under our experimental conditions, about 80% disaccharides (six identified disaccharides with different number and position
F
Slow moving D G
A
I-leparin
II E
11
I
10
20
30
5O
Fast moving E c
w ¢s
n
.L 0
I
I
I
1
20
30
40
I
Retention Time Irnin) Fig. 2. Chromatographic separations of constituent disaccharides of slow moving and fast moving heparin species obtained by cleaving with heparinase I + I I + I I I . (A) ADiH-0S, (B) ADiH-NS, (C) ADiH-6S, (D) ADiH-2,NdiS, (E) ADiH-N,6diS, (F) ADiH-TriS. See abbreviations and Table l for structures of the disaccharides.
J,
1
2
3
a,
5
6
7
Fig. 3. Polyacrylamide-gel electrophoresis of different LMM-heparins compared with unfractionated species (Hep I). 1 M r = 950, 2 M r = 1530, 3 Mr=3210, 4 Mr=4180, 5 Mr=5740, 6 Mr=7820, 7 (Hep I) M~ = 13 100; The arrow indicates the direction of electrophoretic migration.
of sulfate groups, as illustrated in Table 1) and about 20% oligosaccharides still resistant to treatment with heparinases. The disaccharide pattern of heparin consists of about 3.8% unsaturated non-sulfated disaccharide, 4.2% monosulfated disaccharides, about 30.0% disulfated disaccharides and about 62% trisulfated disaccharide (Table 1). This preparation of heparin shows a percentage of N-sulfate groups of about 94% and N-acetyl groups of about 6%, and a percentage of iduronic acid of about 90%, and with remaining 10% glucuronic acid (Table 1). Hep II shows small differences with respect to Hep I. In particular, this bovine mucosa heparin preparation has a smaller percentage of trisulfated disaccharide with a smaller value of sulfate-to-carboxyl ratio (2.45), iduronic acid and N-sulfate group percentages, and anticoagulant activity (155 IU/mg). Physico-chemical properties and unsaturated disaccharide percentages of slow and fast moving heparins are illustrated in Table 1. The fast moving species has a decreased M r, sulfate-to-carboxyl ratio (with a decrease of unsaturated trisulfated disaccharide, see also Fig. 2), Nsulfate groups and iduronic acid percentage with respect to native heparin. As expected, there is also a decrease in anticoagulant activity. On the contrary, the slow moving heparin species shows higher M r, a great amount of trisulfated disaccharide (Fig. 2) and N-sulfate groups, and high values of charge density and anticoagulant activity. The physico-chemical properties of bovine spleen heparan sulfate are reported in Table 1 and compared with that of chemically modified heparin. The Mr of bovine spleen heparan sulfate is about 38 300 with a high polydispersity (from Mr > 50000 to 10000); the Mr of chemically modi-
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N. Volpi / Biochiraica et Biophysica Acta 1290 (1996) 299-307 120-
Table 2 Mr, charge density (determined by potentiometric titration), and anticoagulant activity of LMM-heparin fractions prepared by chemical depolymerization in the presence of free radicals [21,22] Mr (X 1 0 0 0 )
SO~-/CO0-
APTI' (IO/mg)
7,82 5.74 4.18 3.21 1.53 0.95
2.36 2.32 2.34 2.27 2.25 2.25
67 36 21 7 5 <2
r
1-
T
-{
100-
°o
80" ~
HA
60-
•
~ -r
Hep I
4020!
!
5
10 I~g/ml of GAGs
Fig. 4. Human leukocyte elastase activity (% of control) in terms of increasing amounts (from 0.1 to 10 ixg) of Hep I and hyaluronic acid.
fled heparin is about 8700 (M r from 14300 to 3000) due to a partial depolymerization of native heparin under the adopted chemical procedure. Purified heparan sulfate has a large amount of nonsulfated and monosulfated disaccharides (about 66% of total disaccharides) and only 5.7% of trisulfated disaccharide, a sulfate-to-carboxyl ratio of about 0.94 with about 55% of N-sulfate groups and 82% of glucuronic acid. The anticoagulant activity is very low ( < 0.5 IU/mg). The sulfate-to-carboxyl ratio of the partially desulfated and re-N-acetylated heparin is about 1.47 due to the partial O- and N-desulfation of native heparin, with about 33% nonsulfated disaccharide, 18% monosulfated disaccharides and 31% trisulfated disaccharide (Table 1). This heparin derivative has about 60% N-sulfate groups and 40% N-acetyl groups (an increase of about 32.4% with respect to heparin) (Table 1). The percentage of iduronic acid is the same as heparin, and there is a drastic decrease in anticoagulant activity (about 10 IU/mg) due to chemical modification of the primary structure. LMM-heparins with various M r were prepared by chemical process of depolymerization in the presence of free radicals [21,22]. These fractions were further fractionated by gel-permeation chromatography to obtain species with narrow polydispersity with respect to the unfractionated heparin (Fig. 3). Table 2 shows the M r of these heparin derivatives (evaluated by HPSEC) with their charge density values and anticoagulant activities, and Fig. 3 compares the electrophoretic mobilities and polydispersity of these LMM-fractions with that of unfractionated heparin. As previously reported [22], the chemical degradation of heparin by free radicals allows to prepare LMM-derivafives in large amounts with desired M r. These fractions are characterized by a limited decrease in the charge density [22] respect to that of unfractionated species, and this was proved by titrimetric evaluation of sulfate-to-carboxyl ratio (Table 2), by their capacity to bind cationic dye such as 1,9-dimethylmethylene blue and to interact with cationic groups of anion-exchange resins such as ecteola-cellulose (data not shown). The anticoagulant activities, expressed as APTT, of very LMM-heparins decreases in proportion to M r, according to Holmer et al. [32].
100-
~ E
80-
"6
S0.
'>
4.0-
""
20.
00
0"
,~
-
=
g
g
g
=
Q ~0
Z it3 a o.
GAGs (1 I.tg)
Fig. 5. Human leukocyte elastase activity (% of control) in the presence of 1 I~g of hyaluronic acid (HA), heparins (Hep I and Hep II), slow moving (SM Hep) and fast moving heparin species (FM Hep), heparan sulfate (HS) and partially desulfated and re-N-acetylated heparin (PDSNAcHep).
lOO
80-
~ 60> ~ 40::=: 20 =
0 0
!
!
1
2 s03-/c00-
Fig. 6. Relationship between human leukocyte elastase activity (% of control) and charge density of beparins and heparan sulfate.
306
N. Volpi / Biochimica et Biophysica Acta 1290 (1996) 299-307 100 y = -0.823x+ 116.961
Table 3 Effects of heparins (Hep I and Hep I1, 1 i~g) on HLE activity mediated by changes in the ionic strength of the medium
r 2 =0.858
= 80-
HLE activity (% of control) 60-
NaCI (M) Hep I Hep I1
.~_ 4 0 -
~' ao0
50
6'0
7'0
8'0
9.0
1oo
% N-Sulfate groups
Fig. 7. Relationship between human leukocyte elastase activity (% of control) and percentage of N-sulfate groups of heparins and heparan sulfate.
3.2. Effect of heparins, heparan sulfate and derivatices on HLE activity in vitro Fig. 4 illustrates the effect of increasing amount of hyaluronic acid and Hep I (from 0.1 to 10 p~g) on HLE activity. Hyaluronic acid is unable to influence the enzy100 y = -0.527x + 90.510 i
r 2 = 0.850
806o-
~ 40:c 2 0 -
2=0
4=0
6'0
8'0
100
% Iduronic acid
Fig. 8. Relationship between human leukocyte elastase activity (% of control) and iduronic acid percentage of heparins and heparan sulfate. 100-
T
Heparins (lp~g/ml)
80 8
0.05 38 41
0.2 63 69
0.4 85 92
0.6 100 100
0.8 100 100
matic activity whilst Hep 1 inhibits HLE activity and the percentage of inhibition increases depending on heparin concentration. To evaluate the influence of various GAGs, I tested HLE activity in the presence of 0.1 and 1 Ixg of polysaccharides (on 15 mU of HLE). Fig. 5 illustrates the effects of 1 Ixg of GAGs on enzymatic activity. Hep I and Hep II inhibit HLE activity to the same extent, whilst the fast moving component has a lower capacity to inhibit enzymatic activity. On the contrary, the slow moving species produces a greater inhibition of elastase activity. The HLE activity (% of control) was fitted versus the numeric values that quantitatively express the different physico-chemical properties of various GAGs. Fig. 6 depicts the linear dependence of charge density (sulfate-tocarboxyl ratio) on enzymatic activity, expressed as percentage of control. We also found a significant linear correlation between the percentage of N-sulfate groups, the increase of HLE activity inhibition (Fig. 7), and the percentage of iduronic acid and enzymatic activity (Fig. 8). To investigate the effect of M r on HLE activity, heparin fractions with different mass and the same charge density produced by a chemical process of depolymerization in the presence of free radicals [21,22] were tested for their ability to inhibit HLE activity. Fig. 9 demonstrates that heparins with a M r greater than about 2000-3000 inhibit the HLE activity to the same extent whilst the fractions with a M r of 1530 and 950 have less capacity to produce a decrease in the enzymatic activity, Since previous reports suggest that there are electrostatic interactions between anionic groups of GAGs and cationic groups of several lysosomal acid hydrolases [33], this author tested the possibility of blocking the interactions between HLE and heparins by increasing the ionic strength of the buffer in which the enzyme and heparins are incubated. Indeed, Table 3 shows that the inhibition of HLE activity by heparins was effectively blocked by this procedure.
~- 8o >
=
-I-
'TT
40
T
T .
4. Discussion
T 1
20
!
0
1'0
5
5
M r (xlO00)
Fig. 9. Relationship between human leukocyte elastase activity (% of control) and M r of heparin fractions prepared by chemical depolymerization process in the presence of free radicals [2122].
The interaction of GAGs with HLE and the inhibition of its activity depend on the range of pH studied [8]. This is obvious since the interaction between this serine proteinase and polyanions is driven by electrostatic forces depending on the groups that can be protonated or deprotonated (such as arginine residues of HLE) at different pH. We studied the effect of various heparins on HLE in vitro
N. Volpi / Biochimica et Biophysica Acta 1290 (1996) 299-307
at pH 8.0, which agrees with previous reports that inhibition is seen towards a chromogenic substrate at this pH value [12,13]. Previous reports suggest that the interaction between several acid hydrolases [33] and HLE [12,15] with GAGs is of an electrostatic nature. We clearly demonstrate this ionic interaction, since a higher ionic strength of the incubation medium blocks the inhibition of HLE activity by heparins. Heparin is a potent inhibitor of HLE [13] and the inhibitory effect increases along a linear-like function depending on the charge density of the polysaccharide. HLE is a cationic glycoprotein formed by 218 amino-acid residues with 19 arginines that interacts with polyanions. Previous reports described a tight binding, hyperbolic noncompetitive inhibition mechanism of HLE by heparin [12,15], and a higher degree of sulfation is expected to be accompanied by lower K i values. This is confirmed in studies in which HLE activity is inhibited to different extents by GAGs with different structure and charge density with a degree of binding, in general along the series heparin > heparan sulfate > dermatan sulfate > chondroitin sulfate A or C > hyaluronic acid [8]. Redini et al. [12] studied the inhibitory effect of HLE by heparin oligosaccharides obtained by HNO 2 depolymerization. He concluded that O-sulfated compounds exhibit lower K i values as compared with their N-sulfated counterparts of similar M r value, and that the capacity to inhibit HLE is inversely correlated to the chain length of heparin oligosaccharides. In this study we confirm the importance of charge density of heparins and derivatives in the interaction and inhibition of HLE activity. On the other hand, we found a linear relationship between N-sulfate groups and iduronic acid contents and the percentage of HLE inhibition. This because natural heparins or heparan sulfate used in this study have a proportional increase of charge density, N-sulfate groups and iduronic acid amount [34]. We have also correlated M~ and the inhibition of HLE using heparin fractions with narrow polydispersity and the same charge density. We confirm that a minimal degree of M r is a prerequisite to inhibit HLE (such as heparin of M r = 2000-3000), and heparins with M r higher than this minimal value inhibit HLE activity to the same extent.
Acknowledgements Research supported by C.N.R. grants to N.V. References [1] Sinha, S., Watorek, W., Karr, S., Giles, J., Bode, W. and Travis, J. (1987) Proc. Natl. Acad. Sci. USA 84, 2228-2232.
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[2] Starkey, P.M., Barret, A.J. and Burleigh, M.C. (1977) Biochim. Biophys. Acta 483, 386-397. [3] Taylor, J.C. and Mittman, C. (1987) Pulmonary Emphysema and Proteolysis (Jovanovitch, H.B., ed.), Academic Press, New York. [4] Janoff, A. (1978) Neutral Proteases of Human Polymorphonuclear Leukocytes (Havemann, K. and Janoff, A., eds.), Urban and Schwartzenberg, Baltimore, MD. [5] Vered, M., Simon, S., Dearing, R. and Janoff, A. (1988) Exp. Lung Res. 14, 67-83. [6] Simon, S., Vered, M., Rinehart, A. and Janoff, A. (1988) Exp. Lung Res. 14, 85-99. [7] Nadziejko, C. and Finkelstein, I. (1994) Am. J. Respir. Cell Mol. Biol. 11, 103-107. [8] Marossy, K. (1981) Biochim. Biophys. Acta 659, 351-361. [9] Jackson, R.L., Busch, S.J. and Cardin, A.L. (1991) Physiol. Rev. 71, 481-539. [10] Kjelltn, L. and Lindahl, U. (1991) Annu. Rev. Biochem. 60, 443475. [11] Burgess, W.H. and Maciag, T. (1989) Annu. Rev. Biochem. 58, 575-606. [12] Redini, F., Tixier, J.-M., Petitou, M., Choay, J., Robert, L. and Hornebeck, W. (1988) Biochem. J. 252, 515-519. [13] Walsh, R.L., Dillon, T.J., Scicchitano, R. and McLennan, G. (1991) Clin. Sci. 81,341-346. [14] Cad~ne, M., Boudier, C., De Marcillac, G.D. and Bieth, J.G. (1995) J. Biol. Chem. 270, 13204-13209. [15] Baici, A., Diczhazi, C., Neszmelyi, A., Moczar, E. and Hornebeck, W. (1993) Biochem. Pharm. 46, 1545-1549. [16] Poblacion, C.A. and Michelacci, Y.M. (1986) Carbohydr. Res. 147, 87-100. [17] Volpi, N. (1994) Anal. Biochem. 218, 382-391. [18] Takahashi, H.K., Nader, H.B. and Dietrich, C.P. (1985) Arzneim.Forsch. 35, 1620-1623. [19] Volpi, N. (1993) Carbohydr. Res. 247, 263-278. [20] Danishefsky, I., Eiber, H.B. and Can', J.J. (1960) Arch. Biochem. Biophys. 90, 114-121. [21] Volpi, N., Mascellani, G. and Bianchini, P. (1992) Anal. Biochem. 200, 100-107. [22] Volpi, N. (1993) J. Chromatogr. Biomed. Appl. 622, 13-20. [23] Volpi, N. and Bolognani, L. (1993) J. Chromatogr. 630, 390-396. [24] Volpi, N. (1994) Carbohydr. Res. 255, 133-144. [25] Fransson, L.-A., Roden, L. and Spach, M.L. (1968) Anal. Biochem. 21,317-330. [26] Spiro, R.G. (1966) Methods Enzymol. VIII, 3-25. [27] Rice, K.G., RoUink, M.K. and Linhardt, R.J. (1987) Biochem. J. 244, 515-522. [28] Thomson, J.M, and Poller, L. (1985) Blood Coagulation and Haemostasis (Thomson, J.M., ed.), Churchill Livingstone, Edinburgh. [29] Barrowcliffe, T.W. (1989) in Heparin: Structure, Cellular Functions and Clinical Applications (McDuffie, N.M., ed.), Academic Press, New York. [30] Cappelletti, R., Del Rosso, M. and Chiarugi, V.P. (1979) Anal. Biochem. 99, 311-315. [31] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 317-323. [32] Holmer, E., S&terberg, K., Bergqvist, D. and Lindahl, U. (1986) Haemostasis 16 (Suppl. 2), 1-7. [33] Avila, J.L. and Convit, J. (1975) Biochem. J. 152, 57-64. [34] Lane, D.A. and Lindahl, U., eds. (1989) Heparin: Chemical and Biological Properties. Clinical applications, Edward Arnold, London.
Bioch et Biophysica AEta ELSEVIER
Biochimica et Biophysica Acta 1290 (1996) 299-307
Inhibition of human leukocyte elastase activity by heparins: influence of charge density Nicola Volpi * Department of 'Biologia Animale', Chair of Biological Chemistry, Uni~,ersityof Modena, 1-41100Modena, Italy Received 29 February 1996; accepted 26 March 1996
Abstract
Heparins with different structures and physico-chemical properties were evaluated for their capacity to inhibit human leukocyte elastase activity in vitro by using a chromogenic substrate. Heparin from bovine intestinal mucosa and heparan sulfate from bovine spleen were extracted and purified, and their purity, structures, and physico-chemical properties were evaluated. Slow moving and fast moving heparin species were obtained by selective precipitation as barium salt, and partially desulfated and re-N-sulfated heparin was produced by chemical modifications. Heparins with different molecular mass (from 950 to 7820), narrow polydispersity and the same charge density were produced by a chemical depolymerization process in the presence of free radicals, and further gel-permeation chromatography. Heparins strongly inhibit elastase activity, and there is a significant linear dependence between charge density (sulfate-to-carboxyl ratio) and enzymatic activity. We also found a significant linear correlation between the percentage of N-sulfate groups and increased inhibition of elastase activity and between the percentage of iduronic acid and enzymatic activity. Heparin samples with a Mr greater than about 2000-3000 inhibit the HLE activity to the same extent (about 59%) whilst two fractions with a Mr of 1530 (29% inhibition of HLE activity) and 950 (4% inhibition of HLE activity) have less capacity to produce a decrease in the enzymatic activity. Keywords: Elastase; Glycosaminoglycan; Heparin; Heparan sulfate; (Human leukocyte)
1. Introduction
Human leukocyte elastase [EC 3.4.21.37] (HLE), a cationic glycoprotein with p I near 10 and 30 kDa formed by 218 amino-acid residues with 19 arginines [1], is an essential component of polymorphonuclear leukocytes. This serine proteinase has several physiologic functions including proteolysis of phagocytosed proteins and plasma
Abbreviations: HPSEC, high-performance size-exclusion chromatography; Mr, relative molecular mass; SAX-HPLC, strong anion-exchange high-performance liquid chromatography; LMM, low molecular mass; HLE, human leukocyte elastase; GAGs, glycosaminoglycans; SLAPN, N-succinyl-(L-alanine)3-p-nitroanilide; ADiH-0S, 2-acetarnido-2-deoxy-4O-(4-deoxy-ot-L-threo-hex-4-enepyranosyluronic acid)-o-glucose; ADiHNS, 2-deoxy-2-sulfamino-4-O-(4-deoxy-et-L-threo-hex-4-enepyranosyluronic acid)-o-glucose; ADiH-6S, 2-acetamido-2-deoxy-4-O-(4-deoxyet-L-threo-hex-4-enepyrarmsyluronic acid)-6-O-sulfo-D-glucose; ADiH2,NdiS, 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-ct-L-threo-hex-4enepyranosyluronic acid)~D-glucose; ADiH-N,6diS, 2-deoxy-2-sulfamino4-O-(4-deoxy-et-L-threo-hex-4-enepyranosyluronic acid)-6-O-sulfo-D-glucose; ADiH-triS, 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-ot-Lthreo-hex-4-enepyranosyluronic acid)-6-O-sulfo-D-glucose. * Corresponding author. Fax: + 39 59 581020.
proteins (antithrombin III, immunoglobulins and components of the complement system), degradation of connective macromolecular components such as elastin, interstitial and type IV collagens, proteoglycans, laminin, and fibronectin [2,3]. Acute tissue inflammation is associated with the migration and activation of polymorphonuclear neutrophils followed by the release of HLE (present in millimolar amounts in the azurophil granules), which is able to cause tissue damage [3]. An efficient anti-HLE control system is present at inflammatory sites to prevent undesirable degradation of proteins. HLE inhibitors include otl-proteinase inhibitor, ot2-macroglobulin and mucus proteinase inhibitor. The inefficient or defective control of HLE by naturally occurring inhibitors may lead to diseases such as emphysema, rheumatoid arthritis and periodontitis [4]. Due to its strongly cationic nature, human leukocyte elastase binds to and its activity is inhibited by a variety of polyanions, including bacterial polyanions [5], synthetic RNA homopolymers [6], mucus glycoprotein (mucin) [7], and sulfated glycosaminoglycans (GAGs) [8]. Sulfated GAGs, heparin and heparan sulfate, keratan sulfate, and
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chondroitin sulfates, are complex heteropolysaccharides covalently bound to proteins forming proteoglycans localized at extracellular and cellular levels. They interact with several proteins (for reviews see [9-11]), such as proteinase inhibitors, plasma lipoproteins, lipolytic enzymes. growth factors, extracellular matrix proteins, and others. Interaction with proteins and control of their activities determine several physiological (and pharmacological) activities of GAGs, such as anticoagulant, antithrombotic and antiatherosclerotic properties. Purified preparations of heparin (and derivatives such as low molecular mass (LMM)-heparins and oligosaccharides), heparan sulfate, chondroitin sulfate A and C, and dermatan sulfate (chondroitin sulfate B) prepared by extraction and purification from animal tissues, inhibit HLE activity to different extents depending on the structure and physico-chemical properties of GAGs [12,13]. Furthermore, LMM-heparin influences the kinetics of HLE inhibition via a mucus proteinase inhibitor [14]. The use of natural and synthetic (such as N-oleoyl derivatives of heparin) HLE inhibitors has been proposed as a strategic therapy to prevent protein degradation during the healing phase of emphysema or rheumatoid arthritis [15]. Heparin interacts with HLE by a tight binding, hyperbolic, noncompetitive inhibition mechanism [12,15], and a higher degree of sulfation is expected to be accompanied by lower K i values. On the other hand, M r is another key factor that can influence the interaction between heparin and HLE [ 12,13]. We studied the effect of heparin, heparan sulfate and derivatives with different molecular mass (Mr), charge density and chemical properties on HLE activity in an effort to clarify possible structure-function relationships.
2. Materials and m e t h o d s
2.1. Materials Human leukocyte elastase [E.C. 3.4.21.37] (370 units/mg of protein) was obtained from Sigma. Chromogenic substrate N-succinyl-(L-alanine)3-p-nitroanilide (SLAPN) was from Calbiochem. Papain [E.C. 3.4.22.2], trypsin [E.C. 3.4.21.4], collagenase [E.C. 3.4.24.3], DNAse I [E.C. 3.1.21.1] and RNAse A [E.C. 3.1.27.5] were purchased from Sigma. Hyaluronic acid from human umbilical cord was from Sigma. Heparinase I from Flavobacterium heparinum [E.C. 4.2.2.7.] (2000 units/mg of protein; one unit forms 1.0 nanomol of unsaturated uronic acid per min at pH 7.0 at 37°C), Heparinase II from Flavobacterium heparinum [no assigned E.C. number] (47 units/mg of protein; one unit forms 0.1 Ixmol of unsaturated uronic acid per hour at pH 7.0 at 25°C), Heparinase III from Flavobacterium heparinum [E.C. 4.2.2.8] (215 units/mg of protein; one unit forms 0.1 Ixmoi of unsaturated uronic acid per hour at pH 7.0 at 25°C) were
obtained from Sigma. Unsaturated heparin/heparan sulfate disaccharides (ADiH-0S, ADiH-NS, ADiH-6S, ADiH2,NdiS, ADiH-N,6diS and ADiH-triS, see abbreviations) were obtained from Seikagaku (Tokyo, Japan). Diatomite filters (High Performance Filter Aids) were from Dicalite (Los Angeles, CA, USA). Ecteola-cellulose was from Serva (Heidelberg, Germany). Cation-exchange resin Amberlite IR-120 in Na + form and anion-exchange resin Amberlite IRA-400 in OH- form were from Supelco (Bellefonte, USA). Chelex 100 chelating resin was from Bio-Rad (Richmond, CA, USA). Bio-Gel P6 (particle size range, 45-90 txm; molecular mass range from 1000 to 6000) and Bio-Gel P2 (particle size range, 45-90 I~m; molecular mass range from 100 to 1800) were from BioRad (Richmond, CA, USA). Column Protein Pak 125 (300 × 7.8 ram; particle size: 10 txm; molecular mass ranges: native globular from 2000 to 80000 and random coil from 1000 to 30000; cod. 84601) and Protein Pak 300 (300 × 7.5 mm; particle size: 10 p~m; molecular mass ranges: native globular from 10 000 to 400 000 and random coil from 2000 to 150000; cod. T72711) were from Waters (Milford, USA). 10 I~m Spherisorb SAX (trimethylammoniopropyl groups Si-CH2-CH2-CH2-N+(CH3)3 in CI form) was from Phase Separations (Deeside Industrial Park, Deeside Clwyd, UK). Multiphor II electrophoretic cell (from Pharmacia LKB Biotechnology, Uppsala, Sweden) was used for agarose-gel electrophoresis. Polyacrylamide-gel electrophoresis was performed on a Bio-Rad (Richmond, CA, USA) Protean Ilxi vertical-slab-gel unit connected to a Pharmacia-LKB (Pharmacia, Uppsala, Sweden) 2219 Multitemp II thermostatic circulator. Power was supplied by an LKB 2197 power supply. High purity agarose, acrylamide, bisacrylamide, and barium acetate were from Bio-Rad. 1,2-Diaminopropane and cetyltrimethylammonium bromide were from Merck (Darmstadt, Germany). Bromophenol blue and Azure A were from Sigma (St. Louis, MO, USA). The densitometric instrument was composed of a Macintosh Ilsi computer interfaced with Microtek Color Scanner from Microtek (Hsinchu, Taiwan). IMAGEprocessing and analysis program, Ver. 1.41 from the Jet Propulsion Laboratory of NASA (Florida, USA) was used for densitometric analysis of agarose-gel electrophoretic bands. All the other reagents were analytical grade.
2.2. Extraction and purification of heparin Bovine intestinal mucosa was ground and treated with papain at 65°C for 12 hours in a reaction vessel. After heating to 100°C for 30 rain, the mixture was brought to pH 9.0 by adding 2 N NaOH. After 24 h at 40°C, the product (brought to pH 6.0 with 2 N acetic acid) was filtered on a diatomite filter, and the solution containing polysaccharides was percolated through the ecteola-cellulose resin column. Peptides, nucleic acids and oligosaccharides were eluted with 0.2 M NaC1. Heparin was
N. Volpi/ Biochimicaet BiophysicaActa 1290(1996)299-307 eluted with 3.0 M NaCI, and the recovered solution was added to 1.5 vols. of acetone. The precipitate was solubilized in water and any dermatan sulfate present was removed by selective precipitation as its copper salt [16]. Heparin was further purified by sequential precipitation in 0.6 vol. of acetone [17] and transformed into its sodium salt on Amberlite IR-120 cation-exchange resin in Na + form. Crude heparin sodium salt was collected by precipitation with 2.0 vols. of acetone and dried. This preparation of heparin was called Hep I. A sample of purified heparin from bovine intestinal mucosa was a gift of OmniaBios (Brescia, Italy) and called Hep II.
2.3. Purification of fast moving and slow moving heparin species The slow moving and fast moving components of heparin were purified as their barium salts at different temperatures, as previously reported [18,19]. Purified bovine intestinal mucosa heparin was dissolved in water, and barium acetate (5%) was added slowly with stirring (the pH of the solution was adjusted to 6.0-7.0). After heating to 50-70°C, the solution was left at room temperature (2025°C) for 24 h. The precipitate obtained was solubilized in water and transformed into its sodium salt on Amberlite 1R-120 resin. The crude slow moving heparin species sodium salt was collected by precipitation with 2.0 volumes of acetone and dried. The supernatant was maintained at 5°C for 24 h and the precipitate was collected by centrifugation at 5°C. The fast moving species barium salt was purified as reported for slow moving species.
2.4. Extraction and purification of heparan sulfate B o v i n e s p l e e n was g r o u n d in 500 ml chloroform/methanol 2:1 (v/v). After centrifugation at 5000 rpm for 20 min, the pellet was dried and suspended in double distilled water (500 rnl) and treated with proteolytic enzymes; papain (1 g), trypsin (100 mg) and collagenase (100 mg) were incubated at their optimum temperature (60°C for papain and 37°C for the other proteinases) for 24 h in a reaction vessel. After heating at 100°C for 30 min, DNAse I (100 mg) and RNAse A (10 mg) were added, and the mixture was treated at 37°C for 24 h. After heating to 100°C for 30 min, the mixture was brought to pH 9.0 by adding 2 N NaOH. After 24 h at 40°C, the product (brought to pH 6.0 with 2 N acetic acid) was filtered on a diatomite filter, and 1.5 vol. of acetone was added to precipitate heteropolysaccharides. The recovered precipitate was dried at 60°C for 24 h, dissolved in double distilled water and percolated through the ecteola-cellulose resin column. Peptides, proteins, nucleotides and oligosaccharides still present were eluted with 0.2 M NaCI. Heparan sulfate was eluted with 1.8 M NaC1; 1.5 vol. of acetone were added to the recovered solution and the
301
precipitate was dried at 60°C for 24 h. Heparan sulfate was percolated through the Amberlite IR-120 resin and transformed into heparan sulfate sodium salt.
2.5. Chemical modification of heparin Partial desulfation and re-N-acetylation were essentially performed as reported by Danishefsky et al. [20]. Hep I was both N- and O-desulfated by treating for 30 min with 0.2 N HC1 at 70°C. This derivative was re-N-acetylated by adding acetic anhydride (1 ml per 10 g of polysaccharide) at 25-30°C maintaining the pH in the range of 7 - 8 with NaOH. The reaction takes approx. 30 min under agitation. Modified heparin was recovered by precipitation with 1.5 vol. of acetone, dried and transformed to its sodium salt on Amberlite IR- 120 resin.
2.6. Preparation of LMM-heparin fractions by chemical process in the presence of free radicals Different LMM-fractions were obtained from purified heparin (Hep I) by a controlled chemical depolymerization process induced by free radicals in the presence of copper salt, as previously reported [21,22]. Ten grams of heparin and 0.4 g of copper acetate monohydrate (0.02 M) were dissolved in 100 ml of water in a reaction vessel. Temperature was kept at 60°C and the pH adjusted to 7.5 by adding 1 M NaOH solution. A 9% ( v / v ) hydrogen peroxide solution was added at a rate of 10 ml/h. The reaction for heparin was stopped at different times, and at the end of the reaction, the Chelex 100 chelating resin was utilized to remove pollutant copper from the product, and the strong anion-exchange resin Amberlite IRA-400 in O H - form was used to remove acidic pollutants. The pH of the percolate was adjusted to 6.0 by adding excess acetic acid and then two volumes of acetone were added. The precipitate was collected by filtration, washed with acetone and dissolved again in 100 ml of water. One gram of sodium acetate was added to this solution, and then different sodium salt M r fractions were precipitated by 2.0 vols. of acetone. The precipitate was collected and dried. Heparins with different M r were prepared by stopping the chemical depolymerization process at different times. The different molecular mass fractions were further fractionated on a column packed with Bit-Gel P6 (2 × 80 cm) and eluted with 1 M NaC1. The fractions (determined by HPSEC [23]) were desalted on Bit-Gel P2 (1 × 90 cm) and lyophilized.
2.7. Agarose-gel electrophoresis The purity of Hep I and II, slow moving and fast moving heparin species, heparan sulfate and partial desulfated and re-N-acetylated heparin (PDSNAcHep) were analyzed by agarose-gel electrophoresis in barium acetate/1,2-diaminopropane as reported elsewhere [17,19]. The first run was performed in 0.04 M barium acetate buffer (pH 5.8) for 20 min at 60 mA; the second run was
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in 0.05 M 1,2-diaminopropane buffered at pH 9.0 with acetic acid for 45 min at 50 mA. After migration, the plate was soaked in cetyltrimethylammonium bromide (0.1% solution) for about 3 h, dried and then stained with Toluidine blue (0.2% in ethanol/water/acetic acid, 50:49:1 v / v / v ) for 30 min and destained with ethanol/water/ acetic acid 50:49:1 ( v / v / v ) . The slow moving and fast moving component percentages of Hep I and II were analyzed quantitatively by a densitometer and the amounts evaluated by specific calibration curves.
2.8. Determination of peak
Mr
Heparinase I1 and 0.5 units of Heparinase III in 100 mM (pH 7.0) acetate buffer in the presence of 2.5 mmol calcium acetate. The reactions were stopped, after 24 h at 37°C, by boiling 1 min. Constituent disaccharides were determined by SAXHPLC at 232 nm. Isocratic separation was run for 0 to 5 rain with 0.2 M NaCI (pH 4.00); linear gradient separation for 5 to 90 min with 100% 0.2 M NaCI (pH 4.00) to 100% 1.2 M NaC1 (pH 4.00). Flow was 1.4 ml/min. I separated the nonsulfated and variously sulfated disaccharides according to the standards and retention times by Seikagaku Kogyo (see Table 1 and structural formula). The amount of each identified disaccharide was determined by purified standards and reported as weight percent. Under experimental conditions, the simultaneous cleavage of heparin fractions with heparinase I, II and III produces about 75-80% disaccharides and 20-25% oligosaccharides still resistant to treatment with heparinases. Thus, the percentage of each disaccharide was calculated considering the sum of the masses of disaccharides as 100%.
values
HPLC equipment was manufactured by Jasco, Tokyo, Japan (pump mod. 880 PU; system controller mod. 801 SC; ternary gradient unit mod. 880-02; Rheodyne injector equipped with a 100 ~1 loop; UV detector, mod. 875 UV). The mobile phase was composed of 125 mM Na2SO 4 and 2 mM NaH2PO 4 adjusted to pH 6.0 with 0.1 M NaOH. Flow rate was 0.9 ml/min with a back pressure of 25 k g / c m z. Column Protein Pak 125 and Protein Pak 300 were assembled in series. The peak M r (mean value) was determined by a calibration curve plotted with glycosaminoglycans standards, as reported elsewhere [23].
2.10. Determination of sulfate-to-carboxyl ratio Sulfate and carboxyl groups were determined by potentiometric titration [24] with 0.1 N NaOH in water/dimethylformamide of the glycosaminoglycan acid forms obtained by removing cations on the strong exchanger resin Amberlite IR-120 in H + form. The sulfate-to-carboxyl ratio was also determined by enzymatic degradation after HPLC separation of constituent disaccharides. The ratio was calculated by consid-
2.9. Constituent disaccharide quantitation of heparins, heparan sulfate, ,fast moving and slow moving species and partial desulfated and re-N-acetylated heparin 200 ~g (20 m g / m l in H20) of each species were incubated with 18.5 units of Heparinase I, 0.5 units of
Table 1 Disaccharide composition (expressed as weight percent), physico-chemical properties (M r, charge density, uronate percentages, N-sulfate and N-acetyl groups percentages) and anticoagulant activity of purified heparins (Hep I and Hep II), slow moving heparin component (SMHep), fast moving heparin species (FMHep), beef spleen heparan sulfate (HS) and partially desulfated and re-N-acetylated heparin (PDSNAcHep)
ADiH-0S ADiH-NS ADiH-6S A DiH-2,NdiS A DiH-N,6diS A DiH-2,N,6triS M r (X 1000) SO~/COO- ~ NS% ~ NAc% ~ IdoA% ~ GIcA% a APTT ( I U / m g )
RI
R2
R6
COCH~ SO 3 COCH 3 SO~SO~SO~-
H H H SO 3 H SO~-
H H SO3 H SO~SO~-
Hep I
Hep II
3.8 2.2 2.0 I9.0 I 1.0 62.0 13.10 2.50 94.2 5.8 90.0 10.0 170
SMHep
4.3 1.8 2.4 17.2 13.5 60.8 12.35 2.45 93.3 6.7 88.2 I 1.8 155
2.0 0.9 0.2 15.3 10.3 71.2 14.90 2.66 97.5 2.5 95.6 4.4 > 190
FMHep 8.9 5.5 2.5 22.9 22.4 37.7 7.92 2. l 1 88.6 11.4 81.6 18.4 58
HS 21.0 21.1 24.3 11.2 4.7 5.7 38.30 0.94 54.7 45.3 17.7 82.3 < 0.5
PDSNAcHep 33.1 11.2 6.6 10.5 7.0 31.4 8.70 1.47 60.1 39.9 88.2 11.8 10
For structures of the disaccharides see formula:
0;~
H•O•2 OR
~;H20R6 ~
a S 0 3 / C 0 O - ratios are reported as average values from potentiometric and enzymatic determinations; ldoA%, iduronic acid percentage; GlcA%, glucuronic acid percentage; NS%, N-sulfate group percentage; NAc%, N-acetyl group percentage.
N. Volpi / Biochimica et Biophysica Acta 1290 (1996) 299-307
ering the presence and the percentage of carboxyl and sulfate groups for each disaccharide.
2.11. Determination of iduronate / glucuronate ratio Uronate ratio of heparins, fast moving and slow moving species and heparan sulfate was evaluated by repeated acid hydrolysis and deaminative cleavage. The split uronic acids were then separated and quantified by ion-exchange chromatography [25].
2.12. Determination of N-acetyl group of heparins, heparan sulfate and derivatives The amount of N-acetyl groups were determined as reported elsewhere [26] and by separation and quantitation of constituent disaccharides (see Section 2.9).
2.13. Polyacrylamide-gel electrophoresis The gel was prepared essentially as reported by Rice et al. [27]. The resolving gel and lower buffer chamber contained 0.1 M boric acid/0.1 M Tris/0.01 M disodium EDTA buffer (pH 8.3). The stacking gel was prepared in the same buffer adjusted to pH 6.3 with HCI. The upper buffer chamber contained 0.2 M Tris/1.25 M glycine hydrochloride (pH 8.3). Gels were poured vertically, using a gel-pouring stand, between glass plates (20 X 16 cm) separated by 1.0 mm spacers. Gradient gels were poured by adding 20 ml of 12% total acrylamide (acrylamide + bisacrylamide) solution to the front chamber of the gradient apparatus and 20 ml of 25% total acrylamide solution to the back chamber. A 5 ml portion of stacking-gel solution containing 5% total acrylamide was applied to the top of the resolving gel. Various LMM-heparin samples (5-10 p,l, 10 m g / m l of distilled water) were combined with 20 pA of 50% ( w / v ) sucrose solution containing 0.1% ( w / v ) Bromophenol blue. Electrophoresis was performed at 50 mA (400-500 V) for 2.5-3.0 h at 5°C. Gels were removed from the glass plates and stained for 30 min in 0.08% (w/v) Azure A in water. Destaining was accomplished with rinsing.
303
sample (heparin, heparan sulfate or derivatives at different concentration) were mixed with 165 p,1 of 0.05 M Tris-HC1, 0.05 M NaCI (pH 8.0), 15 pA of HLE at a concentration of 1 mU/pA in the same buffer were added, and the solution was incubated for 60 s at 25°C. 20 pA of 5 mM of the chromogenic substrate SLAPN (dissolved in 10% of dimethylsulfoxide in Tris-HC1 buffer) were added and the reaction incubated at 25°C for 24 h. The reaction was stopped by adding 100 pA of 0.5 M acetic acid, and the rate of hydrolysis was monitored at 405 nm versus a blank composed of chromogenic substrate in buffer. The results are expressed as percentage inhibition in comparison with controls (without addition of GAGs). To demonstrate the ionic interactions between enzyme and heparins (Hep I and Hep II at a concentration of 1 p,g), assays were performed with buffer (0.05 M Tris-HC1, pH 8.0) containing increasing ionic strength of NaC1, 0.2 M, 0.4 M, 0.6 M and 0.8 M, and compared with controls composed of 0.05 M Tris-HC1, 0.05 M NaC1, pH 8.0 buffer.
3. Results
3.1. Determination of structures and properties of heparins, heparan sulfate and deriuatives The purity of the various glycosaminoglycans was carefully evaluated by different methods. Electrophoretic separations in agarose-gel (Fig. l) and in Titan III [30] show 'pure' preparations of heparins (Hep I and II), fast moving species, native heparan sulfate and partially desulfated and re-N-acetylated heparin. Heparin is formed by two components [18], a slow and fast moving moiety, and Hep I has about 40% of the first and 60% of the second, whilst Hep II shows about 32% of the most highly sulfated and higher-Mr species and 68% of the less sulfated and lowerM r species.
2.14. Determination of in vitro anticoagulant activity The anticoagulant activity of heparin, fast moving and slow moving species, bovine spleen heparan sulfate, partially desulfated and re-N-acetylated heparin, and different LMM-heparins was determined as Activated Partial Thromboplastin Time activity (APTT activity) [28]. The APTT activity was determined versus the WHO IV International Heparin Standard [29] and expressed as IU/mg.
2.15. HLE chromogenic assay This assay was performed essentially as reported by Walsh et al. [13] with minor modifications. 100 ~1 of
Fig. 1. Agarose-gel electrophoretic separation of glycosaminoglycans. 1 Glycosaminoglycan standards (A - slow moving species, B - fast moving species, C - dermatan sulfate, D - chondroitin sulfate); 2 Hep I, 5 ~.g; 3 Hep II, 5 lug; 4 slow moving heparin, 5 p.g; 5 fast moving heparin, 5 p.g; 6 heparan sulfate, 5 ~,g; and 7 partially desulfated and re-N-acetylated heparin, 5 lug. The arrow indicates the direction of electrophoretic migration.
304
N. Volpi / Biochimica et Biophysica Acta 1290 (1996) 299-307
The slow and fast moving species were prepared by fractionation of heparin barium salt at different temperatures [18,19]. The slow moving heparin preparation has about 7% of the fast moving species whilst the fast moving heparin is composed of 100% pure component, as tested by electrophoresis (Fig. 1). Agarose-gel electrophoresis of bovine spleen heparan sulfate shows an electrophoretic band with mobility similar to fast moving heparin (Fig. 1). Analysis of the constituent disaccharides (see below) demonstrates that the fast moving component of heparin and heparan sulfate are different macromolecules in spite of their similar agarose-gel electrophoretic mobility. The possible presence of peptides or proteins in preparations of heparins and heparan sulfate used in the experiments was tested by the Folin phenol reagent [31], confirming the absence (less than 0.1% w / w ) of peptides or proteins. Hep I has an M r = 13100, with 90% of the heteropolysaccharide chains ranging from M r = 23700 to M r = 6000, a sulfate-to-carboxyl ratio of 2.50 and an anticoagulant activity of 170 IUflmg. The simultaneous cleavage of heparin with heparinase I, II and III produces, under our experimental conditions, about 80% disaccharides (six identified disaccharides with different number and position
F
Slow moving D G
A
I-leparin
II E
11
I
10
20
30
5O
Fast moving E c
w ¢s
n
.L 0
I
I
I
1
20
30
40
I
Retention Time Irnin) Fig. 2. Chromatographic separations of constituent disaccharides of slow moving and fast moving heparin species obtained by cleaving with heparinase I + I I + I I I . (A) ADiH-0S, (B) ADiH-NS, (C) ADiH-6S, (D) ADiH-2,NdiS, (E) ADiH-N,6diS, (F) ADiH-TriS. See abbreviations and Table l for structures of the disaccharides.
J,
1
2
3
a,
5
6
7
Fig. 3. Polyacrylamide-gel electrophoresis of different LMM-heparins compared with unfractionated species (Hep I). 1 M r = 950, 2 M r = 1530, 3 Mr=3210, 4 Mr=4180, 5 Mr=5740, 6 Mr=7820, 7 (Hep I) M~ = 13 100; The arrow indicates the direction of electrophoretic migration.
of sulfate groups, as illustrated in Table 1) and about 20% oligosaccharides still resistant to treatment with heparinases. The disaccharide pattern of heparin consists of about 3.8% unsaturated non-sulfated disaccharide, 4.2% monosulfated disaccharides, about 30.0% disulfated disaccharides and about 62% trisulfated disaccharide (Table 1). This preparation of heparin shows a percentage of N-sulfate groups of about 94% and N-acetyl groups of about 6%, and a percentage of iduronic acid of about 90%, and with remaining 10% glucuronic acid (Table 1). Hep II shows small differences with respect to Hep I. In particular, this bovine mucosa heparin preparation has a smaller percentage of trisulfated disaccharide with a smaller value of sulfate-to-carboxyl ratio (2.45), iduronic acid and N-sulfate group percentages, and anticoagulant activity (155 IU/mg). Physico-chemical properties and unsaturated disaccharide percentages of slow and fast moving heparins are illustrated in Table 1. The fast moving species has a decreased M r, sulfate-to-carboxyl ratio (with a decrease of unsaturated trisulfated disaccharide, see also Fig. 2), Nsulfate groups and iduronic acid percentage with respect to native heparin. As expected, there is also a decrease in anticoagulant activity. On the contrary, the slow moving heparin species shows higher M r, a great amount of trisulfated disaccharide (Fig. 2) and N-sulfate groups, and high values of charge density and anticoagulant activity. The physico-chemical properties of bovine spleen heparan sulfate are reported in Table 1 and compared with that of chemically modified heparin. The Mr of bovine spleen heparan sulfate is about 38 300 with a high polydispersity (from Mr > 50000 to 10000); the Mr of chemically modi-
305
N. Volpi / Biochiraica et Biophysica Acta 1290 (1996) 299-307 120-
Table 2 Mr, charge density (determined by potentiometric titration), and anticoagulant activity of LMM-heparin fractions prepared by chemical depolymerization in the presence of free radicals [21,22] Mr (X 1 0 0 0 )
SO~-/CO0-
APTI' (IO/mg)
7,82 5.74 4.18 3.21 1.53 0.95
2.36 2.32 2.34 2.27 2.25 2.25
67 36 21 7 5 <2
r
1-
T
-{
100-
°o
80" ~
HA
60-
•
~ -r
Hep I
4020!
!
5
10 I~g/ml of GAGs
Fig. 4. Human leukocyte elastase activity (% of control) in terms of increasing amounts (from 0.1 to 10 ixg) of Hep I and hyaluronic acid.
fled heparin is about 8700 (M r from 14300 to 3000) due to a partial depolymerization of native heparin under the adopted chemical procedure. Purified heparan sulfate has a large amount of nonsulfated and monosulfated disaccharides (about 66% of total disaccharides) and only 5.7% of trisulfated disaccharide, a sulfate-to-carboxyl ratio of about 0.94 with about 55% of N-sulfate groups and 82% of glucuronic acid. The anticoagulant activity is very low ( < 0.5 IU/mg). The sulfate-to-carboxyl ratio of the partially desulfated and re-N-acetylated heparin is about 1.47 due to the partial O- and N-desulfation of native heparin, with about 33% nonsulfated disaccharide, 18% monosulfated disaccharides and 31% trisulfated disaccharide (Table 1). This heparin derivative has about 60% N-sulfate groups and 40% N-acetyl groups (an increase of about 32.4% with respect to heparin) (Table 1). The percentage of iduronic acid is the same as heparin, and there is a drastic decrease in anticoagulant activity (about 10 IU/mg) due to chemical modification of the primary structure. LMM-heparins with various M r were prepared by chemical process of depolymerization in the presence of free radicals [21,22]. These fractions were further fractionated by gel-permeation chromatography to obtain species with narrow polydispersity with respect to the unfractionated heparin (Fig. 3). Table 2 shows the M r of these heparin derivatives (evaluated by HPSEC) with their charge density values and anticoagulant activities, and Fig. 3 compares the electrophoretic mobilities and polydispersity of these LMM-fractions with that of unfractionated heparin. As previously reported [22], the chemical degradation of heparin by free radicals allows to prepare LMM-derivafives in large amounts with desired M r. These fractions are characterized by a limited decrease in the charge density [22] respect to that of unfractionated species, and this was proved by titrimetric evaluation of sulfate-to-carboxyl ratio (Table 2), by their capacity to bind cationic dye such as 1,9-dimethylmethylene blue and to interact with cationic groups of anion-exchange resins such as ecteola-cellulose (data not shown). The anticoagulant activities, expressed as APTT, of very LMM-heparins decreases in proportion to M r, according to Holmer et al. [32].
100-
~ E
80-
"6
S0.
'>
4.0-
""
20.
00
0"
,~
-
=
g
g
g
=
Q ~0
Z it3 a o.
GAGs (1 I.tg)
Fig. 5. Human leukocyte elastase activity (% of control) in the presence of 1 I~g of hyaluronic acid (HA), heparins (Hep I and Hep II), slow moving (SM Hep) and fast moving heparin species (FM Hep), heparan sulfate (HS) and partially desulfated and re-N-acetylated heparin (PDSNAcHep).
lOO
80-
~ 60> ~ 40::=: 20 =
0 0
!
!
1
2 s03-/c00-
Fig. 6. Relationship between human leukocyte elastase activity (% of control) and charge density of beparins and heparan sulfate.
306
N. Volpi / Biochimica et Biophysica Acta 1290 (1996) 299-307 100 y = -0.823x+ 116.961
Table 3 Effects of heparins (Hep I and Hep I1, 1 i~g) on HLE activity mediated by changes in the ionic strength of the medium
r 2 =0.858
= 80-
HLE activity (% of control) 60-
NaCI (M) Hep I Hep I1
.~_ 4 0 -
~' ao0
50
6'0
7'0
8'0
9.0
1oo
% N-Sulfate groups
Fig. 7. Relationship between human leukocyte elastase activity (% of control) and percentage of N-sulfate groups of heparins and heparan sulfate.
3.2. Effect of heparins, heparan sulfate and derivatices on HLE activity in vitro Fig. 4 illustrates the effect of increasing amount of hyaluronic acid and Hep I (from 0.1 to 10 p~g) on HLE activity. Hyaluronic acid is unable to influence the enzy100 y = -0.527x + 90.510 i
r 2 = 0.850
806o-
~ 40:c 2 0 -
2=0
4=0
6'0
8'0
100
% Iduronic acid
Fig. 8. Relationship between human leukocyte elastase activity (% of control) and iduronic acid percentage of heparins and heparan sulfate. 100-
T
Heparins (lp~g/ml)
80 8
0.05 38 41
0.2 63 69
0.4 85 92
0.6 100 100
0.8 100 100
matic activity whilst Hep 1 inhibits HLE activity and the percentage of inhibition increases depending on heparin concentration. To evaluate the influence of various GAGs, I tested HLE activity in the presence of 0.1 and 1 Ixg of polysaccharides (on 15 mU of HLE). Fig. 5 illustrates the effects of 1 Ixg of GAGs on enzymatic activity. Hep I and Hep II inhibit HLE activity to the same extent, whilst the fast moving component has a lower capacity to inhibit enzymatic activity. On the contrary, the slow moving species produces a greater inhibition of elastase activity. The HLE activity (% of control) was fitted versus the numeric values that quantitatively express the different physico-chemical properties of various GAGs. Fig. 6 depicts the linear dependence of charge density (sulfate-tocarboxyl ratio) on enzymatic activity, expressed as percentage of control. We also found a significant linear correlation between the percentage of N-sulfate groups, the increase of HLE activity inhibition (Fig. 7), and the percentage of iduronic acid and enzymatic activity (Fig. 8). To investigate the effect of M r on HLE activity, heparin fractions with different mass and the same charge density produced by a chemical process of depolymerization in the presence of free radicals [21,22] were tested for their ability to inhibit HLE activity. Fig. 9 demonstrates that heparins with a M r greater than about 2000-3000 inhibit the HLE activity to the same extent whilst the fractions with a M r of 1530 and 950 have less capacity to produce a decrease in the enzymatic activity, Since previous reports suggest that there are electrostatic interactions between anionic groups of GAGs and cationic groups of several lysosomal acid hydrolases [33], this author tested the possibility of blocking the interactions between HLE and heparins by increasing the ionic strength of the buffer in which the enzyme and heparins are incubated. Indeed, Table 3 shows that the inhibition of HLE activity by heparins was effectively blocked by this procedure.
~- 8o >
=
-I-
'TT
40
T
T .
4. Discussion
T 1
20
!
0
1'0
5
5
M r (xlO00)
Fig. 9. Relationship between human leukocyte elastase activity (% of control) and M r of heparin fractions prepared by chemical depolymerization process in the presence of free radicals [2122].
The interaction of GAGs with HLE and the inhibition of its activity depend on the range of pH studied [8]. This is obvious since the interaction between this serine proteinase and polyanions is driven by electrostatic forces depending on the groups that can be protonated or deprotonated (such as arginine residues of HLE) at different pH. We studied the effect of various heparins on HLE in vitro
N. Volpi / Biochimica et Biophysica Acta 1290 (1996) 299-307
at pH 8.0, which agrees with previous reports that inhibition is seen towards a chromogenic substrate at this pH value [12,13]. Previous reports suggest that the interaction between several acid hydrolases [33] and HLE [12,15] with GAGs is of an electrostatic nature. We clearly demonstrate this ionic interaction, since a higher ionic strength of the incubation medium blocks the inhibition of HLE activity by heparins. Heparin is a potent inhibitor of HLE [13] and the inhibitory effect increases along a linear-like function depending on the charge density of the polysaccharide. HLE is a cationic glycoprotein formed by 218 amino-acid residues with 19 arginines that interacts with polyanions. Previous reports described a tight binding, hyperbolic noncompetitive inhibition mechanism of HLE by heparin [12,15], and a higher degree of sulfation is expected to be accompanied by lower K i values. This is confirmed in studies in which HLE activity is inhibited to different extents by GAGs with different structure and charge density with a degree of binding, in general along the series heparin > heparan sulfate > dermatan sulfate > chondroitin sulfate A or C > hyaluronic acid [8]. Redini et al. [12] studied the inhibitory effect of HLE by heparin oligosaccharides obtained by HNO 2 depolymerization. He concluded that O-sulfated compounds exhibit lower K i values as compared with their N-sulfated counterparts of similar M r value, and that the capacity to inhibit HLE is inversely correlated to the chain length of heparin oligosaccharides. In this study we confirm the importance of charge density of heparins and derivatives in the interaction and inhibition of HLE activity. On the other hand, we found a linear relationship between N-sulfate groups and iduronic acid contents and the percentage of HLE inhibition. This because natural heparins or heparan sulfate used in this study have a proportional increase of charge density, N-sulfate groups and iduronic acid amount [34]. We have also correlated M~ and the inhibition of HLE using heparin fractions with narrow polydispersity and the same charge density. We confirm that a minimal degree of M r is a prerequisite to inhibit HLE (such as heparin of M r = 2000-3000), and heparins with M r higher than this minimal value inhibit HLE activity to the same extent.
Acknowledgements Research supported by C.N.R. grants to N.V. References [1] Sinha, S., Watorek, W., Karr, S., Giles, J., Bode, W. and Travis, J. (1987) Proc. Natl. Acad. Sci. USA 84, 2228-2232.
307
[2] Starkey, P.M., Barret, A.J. and Burleigh, M.C. (1977) Biochim. Biophys. Acta 483, 386-397. [3] Taylor, J.C. and Mittman, C. (1987) Pulmonary Emphysema and Proteolysis (Jovanovitch, H.B., ed.), Academic Press, New York. [4] Janoff, A. (1978) Neutral Proteases of Human Polymorphonuclear Leukocytes (Havemann, K. and Janoff, A., eds.), Urban and Schwartzenberg, Baltimore, MD. [5] Vered, M., Simon, S., Dearing, R. and Janoff, A. (1988) Exp. Lung Res. 14, 67-83. [6] Simon, S., Vered, M., Rinehart, A. and Janoff, A. (1988) Exp. Lung Res. 14, 85-99. [7] Nadziejko, C. and Finkelstein, I. (1994) Am. J. Respir. Cell Mol. Biol. 11, 103-107. [8] Marossy, K. (1981) Biochim. Biophys. Acta 659, 351-361. [9] Jackson, R.L., Busch, S.J. and Cardin, A.L. (1991) Physiol. Rev. 71, 481-539. [10] Kjelltn, L. and Lindahl, U. (1991) Annu. Rev. Biochem. 60, 443475. [11] Burgess, W.H. and Maciag, T. (1989) Annu. Rev. Biochem. 58, 575-606. [12] Redini, F., Tixier, J.-M., Petitou, M., Choay, J., Robert, L. and Hornebeck, W. (1988) Biochem. J. 252, 515-519. [13] Walsh, R.L., Dillon, T.J., Scicchitano, R. and McLennan, G. (1991) Clin. Sci. 81,341-346. [14] Cad~ne, M., Boudier, C., De Marcillac, G.D. and Bieth, J.G. (1995) J. Biol. Chem. 270, 13204-13209. [15] Baici, A., Diczhazi, C., Neszmelyi, A., Moczar, E. and Hornebeck, W. (1993) Biochem. Pharm. 46, 1545-1549. [16] Poblacion, C.A. and Michelacci, Y.M. (1986) Carbohydr. Res. 147, 87-100. [17] Volpi, N. (1994) Anal. Biochem. 218, 382-391. [18] Takahashi, H.K., Nader, H.B. and Dietrich, C.P. (1985) Arzneim.Forsch. 35, 1620-1623. [19] Volpi, N. (1993) Carbohydr. Res. 247, 263-278. [20] Danishefsky, I., Eiber, H.B. and Can', J.J. (1960) Arch. Biochem. Biophys. 90, 114-121. [21] Volpi, N., Mascellani, G. and Bianchini, P. (1992) Anal. Biochem. 200, 100-107. [22] Volpi, N. (1993) J. Chromatogr. Biomed. Appl. 622, 13-20. [23] Volpi, N. and Bolognani, L. (1993) J. Chromatogr. 630, 390-396. [24] Volpi, N. (1994) Carbohydr. Res. 255, 133-144. [25] Fransson, L.-A., Roden, L. and Spach, M.L. (1968) Anal. Biochem. 21,317-330. [26] Spiro, R.G. (1966) Methods Enzymol. VIII, 3-25. [27] Rice, K.G., RoUink, M.K. and Linhardt, R.J. (1987) Biochem. J. 244, 515-522. [28] Thomson, J.M, and Poller, L. (1985) Blood Coagulation and Haemostasis (Thomson, J.M., ed.), Churchill Livingstone, Edinburgh. [29] Barrowcliffe, T.W. (1989) in Heparin: Structure, Cellular Functions and Clinical Applications (McDuffie, N.M., ed.), Academic Press, New York. [30] Cappelletti, R., Del Rosso, M. and Chiarugi, V.P. (1979) Anal. Biochem. 99, 311-315. [31] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 317-323. [32] Holmer, E., S&terberg, K., Bergqvist, D. and Lindahl, U. (1986) Haemostasis 16 (Suppl. 2), 1-7. [33] Avila, J.L. and Convit, J. (1975) Biochem. J. 152, 57-64. [34] Lane, D.A. and Lindahl, U., eds. (1989) Heparin: Chemical and Biological Properties. Clinical applications, Edward Arnold, London.