Biochimica et Biophysica Acta, 439 (1976) 380-392
~" Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
BBA 37402 STUDIES ON PIG S E R U M L I P O P R O T E I N S 111. A F F I N I T Y C H R O M A T O G R A P H Y C O N C A N A V A L I N A-SEPHAROSE
OF
NATIVE
L I P O P R O T E I N S ON
JUN-ICH1 AZUMA, NAOKI KASHIMURA and TOHRU KOMANO Laboratory of Biochemistry, Department of Agricultural Chemistry, Kyoto University, Kyoto (Japan)
(Received January 23rd, 1976)
SUMMARY The comparison of the binding capacities of the three major classes of pig serum lipoproteins, very low-density, low-density and high-density lipoproteins, to concanavalin A, was demonstrated by affinity chromatography on concanavalin A-Sepharose. Very low-density lipoprotein was separated into two fractions (60 to 6 6 ~ of total protein was adsorbed). Each fraction had different electrophoretic mobility in pore size gradient gel. The majority of the carbohydrate was found in the adsorbed fraction. The carbohydrate content of the unadsorbed fraction was 0.14 ~o sialic acid. 0.47 ~ hexosamine and 0.93 ~ neutral sugars, and of the adsorbed fraction~ 2.05, 3.21 and 4.20~, respectively. The adsorbed and unadsorbed fractions contained fucose, mannose and galactose in the molar ratio of 1.0 : 3.6 ± 0.2 : 2.2 ~- 0.4 and 1.0 : 3.1 ~ 0.2 : 2.5 ~ 0.3, respectively. Based on these results, two different molecular species were proved to be present in very low-density lipoproteins. In highdensity lipoproteins, 80 to 85 ~ of the total protein was not adsorbed on concanavalin A-Sepharose in spite of the presence of mannose in the apoprotein. In contrast to these lipoproteins, low-density lipoprotein was completely adsorbed on concanavalin A-Sepharose. However, the separation of the subfractions of low-density lipoprotein as well as the subfractions of high-density lipoprotein could not be achieved by this affinity column. The carbohydrate content of eluted fractions of low-density and high-density lipoproteins was identical with the previously reported values obtained in native lipoproteins. This difference in affinities for concanavalin A was also evidenced by gel electrophoretic profiles in urea and in sodium dodecyl sulfate which showed different glycoprotein distribution in each class of lipoproteins.
INTRODUCTION Pig serum contains three major classes of lipoproteins, the very low-density Abbreviations: VLDL, very low-density lipoprotein of density ~ 1.007 g/ml ; LDL, low-density lipoprotein of density 1.007-1.090 g/ml ; HDL, high-density lipoprotein of density 1.090-1.17 g/ml; apoVLDL, lipid-free protein from VLDL; apoLDL, lipid-free protein from LDL; apoHDL, lipidfree protein from HDL; SDS, sodium dodecyl sulfate.
381 (VLDL), low-density (LDL) and high-density (HDL) lipoproteins [1]. Total LDL has been shown to be ultracentrifugally and electrophoretically heterogeneous and comprize two distinct subfractions, LDL~ and LDL2 [1-4]. HDL is ultracentrifugally homogeneous, but this lipoprotein is electrophoretically heterogeneous, comprizing two subfractions, HDL~ and HDLz [5]. Studies on the apoproteins of the pig serum lipoproteins have been reported by several investigators [6-12]. However, these investigators have not referred to the carbohydrate moiety, although the importance of carbohydrate is strictly emphasized in serum lipoprotein metabolism [13, 14] and in the lipid-protein interaction [15]. In order to elucidate the role of carbohydrate in the structure of lipoproteins, it is essential to separate glycoproteins from nonglycosylated proteins. Recently affinity chromatography has been used extensively for the purification of glycoproteins which are adsorbed specifically on immobilized lectins [16-20]. The use of insoluble forms of concanavalin A prepared by coupling agarose beads has proved a useful method for fractionating blood group substances [17], serum glycoproteins [18], virus induced antigens [19] and membrane glycoproteins [20]. Although this method has been applied to the separation of human plasma lipoproteins [21, 22], the binding capacities of pig serum lipoproteins to concanavalin A have not yet been studied. This communication describes the fractionation of pig serum lipoproteins using affinity chromatography on concanavalin A-Sepharose on the basis of the difference in carbohydrate moiety, and the comparison of the electrophoretic and chemical properties of the native and fractionated lipoproteins. MATERIALS AND METHODS
Materials Concanavalin A-Sepharose 4B prepared by the cyanogen bromide method [18] and Sepharose 4B were supplied by Pharmacia Fine Chemicals. Sodium dodecyl sulfate was purchased from Wako Pure Chem. In. Ltd. Carboxypeptidase A and methyl a-D-mannopyranoside were purchased from Sigma Chemical Co. Etd. Nonenzymatic protein molecular weight markers were obtained from Boehringer Mannheim. All other reagents used were analytical reagent grade.
Preparation of lipoproteins Pig serum lipoprotein classes (VLDL, total LDL and HDL) were prepared by successive ultracentrifugal flotation according to Janado et al. [1] with slight modification [3]. VEDL (d
~" Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
BBA 37402 STUDIES ON PIG S E R U M L I P O P R O T E I N S 111. A F F I N I T Y C H R O M A T O G R A P H Y C O N C A N A V A L I N A-SEPHAROSE
OF
NATIVE
L I P O P R O T E I N S ON
JUN-ICH1 AZUMA, NAOKI KASHIMURA and TOHRU KOMANO Laboratory of Biochemistry, Department of Agricultural Chemistry, Kyoto University, Kyoto (Japan)
(Received January 23rd, 1976)
SUMMARY The comparison of the binding capacities of the three major classes of pig serum lipoproteins, very low-density, low-density and high-density lipoproteins, to concanavalin A, was demonstrated by affinity chromatography on concanavalin A-Sepharose. Very low-density lipoprotein was separated into two fractions (60 to 6 6 ~ of total protein was adsorbed). Each fraction had different electrophoretic mobility in pore size gradient gel. The majority of the carbohydrate was found in the adsorbed fraction. The carbohydrate content of the unadsorbed fraction was 0.14 ~o sialic acid. 0.47 ~ hexosamine and 0.93 ~ neutral sugars, and of the adsorbed fraction~ 2.05, 3.21 and 4.20~, respectively. The adsorbed and unadsorbed fractions contained fucose, mannose and galactose in the molar ratio of 1.0 : 3.6 ± 0.2 : 2.2 ~- 0.4 and 1.0 : 3.1 ~ 0.2 : 2.5 ~ 0.3, respectively. Based on these results, two different molecular species were proved to be present in very low-density lipoproteins. In highdensity lipoproteins, 80 to 85 ~ of the total protein was not adsorbed on concanavalin A-Sepharose in spite of the presence of mannose in the apoprotein. In contrast to these lipoproteins, low-density lipoprotein was completely adsorbed on concanavalin A-Sepharose. However, the separation of the subfractions of low-density lipoprotein as well as the subfractions of high-density lipoprotein could not be achieved by this affinity column. The carbohydrate content of eluted fractions of low-density and high-density lipoproteins was identical with the previously reported values obtained in native lipoproteins. This difference in affinities for concanavalin A was also evidenced by gel electrophoretic profiles in urea and in sodium dodecyl sulfate which showed different glycoprotein distribution in each class of lipoproteins.
INTRODUCTION Pig serum contains three major classes of lipoproteins, the very low-density Abbreviations: VLDL, very low-density lipoprotein of density ~ 1.007 g/ml ; LDL, low-density lipoprotein of density 1.007-1.090 g/ml ; HDL, high-density lipoprotein of density 1.090-1.17 g/ml; apoVLDL, lipid-free protein from VLDL; apoLDL, lipid-free protein from LDL; apoHDL, lipidfree protein from HDL; SDS, sodium dodecyl sulfate.
381 (VLDL), low-density (LDL) and high-density (HDL) lipoproteins [1]. Total LDL has been shown to be ultracentrifugally and electrophoretically heterogeneous and comprize two distinct subfractions, LDL~ and LDL2 [1-4]. HDL is ultracentrifugally homogeneous, but this lipoprotein is electrophoretically heterogeneous, comprizing two subfractions, HDL~ and HDLz [5]. Studies on the apoproteins of the pig serum lipoproteins have been reported by several investigators [6-12]. However, these investigators have not referred to the carbohydrate moiety, although the importance of carbohydrate is strictly emphasized in serum lipoprotein metabolism [13, 14] and in the lipid-protein interaction [15]. In order to elucidate the role of carbohydrate in the structure of lipoproteins, it is essential to separate glycoproteins from nonglycosylated proteins. Recently affinity chromatography has been used extensively for the purification of glycoproteins which are adsorbed specifically on immobilized lectins [16-20]. The use of insoluble forms of concanavalin A prepared by coupling agarose beads has proved a useful method for fractionating blood group substances [17], serum glycoproteins [18], virus induced antigens [19] and membrane glycoproteins [20]. Although this method has been applied to the separation of human plasma lipoproteins [21, 22], the binding capacities of pig serum lipoproteins to concanavalin A have not yet been studied. This communication describes the fractionation of pig serum lipoproteins using affinity chromatography on concanavalin A-Sepharose on the basis of the difference in carbohydrate moiety, and the comparison of the electrophoretic and chemical properties of the native and fractionated lipoproteins. MATERIALS AND METHODS
Materials Concanavalin A-Sepharose 4B prepared by the cyanogen bromide method [18] and Sepharose 4B were supplied by Pharmacia Fine Chemicals. Sodium dodecyl sulfate was purchased from Wako Pure Chem. In. Ltd. Carboxypeptidase A and methyl a-D-mannopyranoside were purchased from Sigma Chemical Co. Etd. Nonenzymatic protein molecular weight markers were obtained from Boehringer Mannheim. All other reagents used were analytical reagent grade.
Preparation of lipoproteins Pig serum lipoprotein classes (VLDL, total LDL and HDL) were prepared by successive ultracentrifugal flotation according to Janado et al. [1] with slight modification [3]. VEDL (d
