2, pp. 473-479, 1992 v
OF UNSATURATED DI CLEAVAGE OF HEPARI LFATE WITH HEPARITI
RIDES BY HEPARAN
K. YOSHIDA,K. MORIKAWA,A. TAWA H. KIKUCHI and K. TOKUYASU Tokyo Research Institute, Seikagaku Corporation, Higashiyyamato, Tok Fax: 0425-63-5848)
~ONO,
(Tel. 0425 63-5811;
(Received 24 February 1992; acce~~ted 25 Mal
Abstract--1. Six kinds of unsaturated disaccharides were prepared pt by ¢ heparan sulfate with heparitinases I0 and IV, and subsequent subse colu identified by HPLC showing good separation from each other. 2. The content of each unsaturated disaccharide fraction fractior was deten to range from 130.7 to 722.3 #reel. 3. Molecular extinction coefficient of each unsaturated disaccharidc at a wavelength of around 230 nm where a peak appeared mated on or the ultravi solution at pH 2. The values varied from 6000 to 6600.
INTRODUCTION
Abbreviations--ADiHS-OS,
I
i
.
.
.
.
.
.
.
.
.
,'trieally, and found d from absorbane¢ ff each disaccharide
wide: Jely. In fact, only 1% of total HS H extracted from bovine kidney possessed a high affinity to antithrot thrombin III in vitro (Yoshida et al.,,1983), indicating that only limited type(s) of HS t had such specific funcl function. It is probable that there is i a unique structure for each binding site of vario~ various types of HS in asso¢ association with its physiological ffunction. Accordingly ingly, it seems necessary to analyze the structures of various types of HS in order to tc distinguish the diffel ~rences between them, mainly by HPLC technique after affording good separation of unsaturated unsa disaecharides produced by heparitinase dige., estion of the target HS polymer. The present study was underta[ aken against these backgrounds, adopting various chromatographic techniques, including HPLC, which afford good separation of unsaturated disacchariddes from HS and heparin (Hep). This paper deals wit] with the preparation and quantitation of unsaturated di disaccharides from HS and Hep, along with the development de~ of an HPLC system by which the unsaturaated disaccharides extinction can be analyzed. In addition, molecular mol coefficients of every unsaturated ddisaccharide were obtained and compared with each other.
Ph 'hysiological functions of heparan sulfate (HS) have been een progressively revealed by accumulation of available ble information concerning the interactions between HS IS and the following physiologically active factors, Itt is known that some types of HS are localized, as heeparan sulfate proteoglycan forms, on the interior surface arface of blood vessels and are capable of attaching themselv aemselves to lipoprotein lipases (Shimada et al., 1981 981,Cheng el a!.,1981),antithrombin III (Marcu m and Rosenberg, 1984, 1985)) and thrombin (Shimada and Ozawa, 1985). Anotherr HS originating in'fibreblast, as a form of beparan sulfate proteoglycan, has the ability to bind fibroblast:growth factor (Flaumenhalt et al., 1990; Kiefer et al.l., 1990; Rapraeger et al., 1991). Because HS has man,ly variations with respect to its localization and origina at at tissue levels, the roles of various types of HS have ,e been assumed to differ auld be addressed. *To whom correspondence should !-acetamido-2-deoxy-4-O-(42-acetamK tmido-2mnosyluronic acid)-o-gludeoxy
OF UNSATURATED DI CLEAVAGE OF HEPARI LFATE WITH HEPARITI
RIDES BY HEPARAN
K. YOSHIDA,K. MORIKAWA,A. TAWA H. KIKUCHI and K. TOKUYASU Tokyo Research Institute, Seikagaku Corporation, Higashiyyamato, Tok Fax: 0425-63-5848)
~ONO,
(Tel. 0425 63-5811;
(Received 24 February 1992; acce~~ted 25 Mal
Abstract--1. Six kinds of unsaturated disaccharides were prepared pt by ¢ heparan sulfate with heparitinases I0 and IV, and subsequent subse colu identified by HPLC showing good separation from each other. 2. The content of each unsaturated disaccharide fraction fractior was deten to range from 130.7 to 722.3 #reel. 3. Molecular extinction coefficient of each unsaturated disaccharidc at a wavelength of around 230 nm where a peak appeared mated on or the ultravi solution at pH 2. The values varied from 6000 to 6600.
INTRODUCTION
Abbreviations--ADiHS-OS,
I
i
.
.
.
.
.
.
.
.
.
,'trieally, and found d from absorbane¢ ff each disaccharide
wide: Jely. In fact, only 1% of total HS H extracted from bovine kidney possessed a high affinity to antithrot thrombin III in vitro (Yoshida et al.,,1983), indicating that only limited type(s) of HS t had such specific funcl function. It is probable that there is i a unique structure for each binding site of vario~ various types of HS in asso¢ association with its physiological ffunction. Accordingly ingly, it seems necessary to analyze the structures of various types of HS in order to tc distinguish the diffel ~rences between them, mainly by HPLC technique after affording good separation of unsaturated unsa disaecharides produced by heparitinase dige., estion of the target HS polymer. The present study was underta[ aken against these backgrounds, adopting various chromatographic techniques, including HPLC, which afford good separation of unsaturated disacchariddes from HS and heparin (Hep). This paper deals wit] with the preparation and quantitation of unsaturated di disaccharides from HS and Hep, along with the development de~ of an HPLC system by which the unsaturaated disaccharides extinction can be analyzed. In addition, molecular mol coefficients of every unsaturated ddisaccharide were obtained and compared with each other.
Ph 'hysiological functions of heparan sulfate (HS) have been een progressively revealed by accumulation of available ble information concerning the interactions between HS IS and the following physiologically active factors, Itt is known that some types of HS are localized, as heeparan sulfate proteoglycan forms, on the interior surface arface of blood vessels and are capable of attaching themselv aemselves to lipoprotein lipases (Shimada et al., 1981 981,Cheng el a!.,1981),antithrombin III (Marcu m and Rosenberg, 1984, 1985)) and thrombin (Shimada and Ozawa, 1985). Anotherr HS originating in'fibreblast, as a form of beparan sulfate proteoglycan, has the ability to bind fibroblast:growth factor (Flaumenhalt et al., 1990; Kiefer et al.l., 1990; Rapraeger et al., 1991). Because HS has man,ly variations with respect to its localization and origina at at tissue levels, the roles of various types of HS have ,e been assumed to differ auld be addressed. *To whom correspondence should !-acetamido-2-deoxy-4-O-(42-acetamK tmido-2mnosyluronic acid)-o-gludeoxy
