Isolation of Human Serum Alpha-2 HS Glycoprotein

Alpha-2 HS glycoprotein was obtained in an almost pure state from normal human serum, by ion exchange chromatography followed by curtain electrophoresis, gel filtration and ammonium sulfate precipitation. Alpha-2 HS glycoprotein has a sedimentation coefficient of 3.5 S, and an approximate molecular weight of 49,000 Dal tons. Purified α₂HS glycoprotein enhances phagocytosis and may be the human homologue of rat α₂ opsonic protein.     

Fast Purification of Phaseolus Vulgaris Isolectins

Abstract

Phytohemagglutinin from red kidney bean has been purified by affinity chromatography on a human α₁-acid glycoprotein Sepha-rose 4B column. Further purification of the hemagglutinin's five isolectins was achieved on a Mono S column with an 86% protein recovery. Each sequentially eluted isolectin from the ion exchange column displayed either hemagglutinating or mitogenic activity. The main activity of each fraction was the result of the combination of varying proportions of the L and E subunits.     

Extracellular α Galactosidase (E.C. 3.2.1.22) from Aspergillus Ficuum NRRL 3135 Purification and Characterization

Abstract

Extracellular α-galactosidase, a glycoprotein from the extracellular culture fluid of Aspergillus ficuum grown on glucose and raffinose in a batch culture system, was purified to homogeneity in five steps by inn exchange and hydrophobic Interaction chromatography. The molecular mass of the enzyme was 70.8 Kd by SDS polyacrylamide gel electrophoresis and 74.1 Kd by gel permeation HPLC. On the basis of a molecular mass of 70.7 Kd, the molar extinction coefficient of the enzyme at 279 nm was estimated to be 6.1 × 10⁴ M^?1 cm^?1. The purified enzyme was remarkably stable at 0°C. It had a broad temperature optimum and maximum catalytic activity was at 60°C. It retained 33% of its activity after 10 min. at 65°C. It had a pH optimum of 6.0. It retained 62% of its activity after 12 hours at pH 2.3. The K_ms for p-nitrophenyl-α-D-galactopyranoside, o-nitrophenyl-α-D-galactopyranoside and m-nitrophenyl-α-D-galactopyranoside are: 1462, 839 and 718 μ. The enzyme was competitively inhibited by mercury (19.8 μ), silver (21.5μM), copper (0.48 mM), zinc (0.11 mM), galactose (64.0 mM) and fructose (60.3 mM). It was inhibited non-competitively by glucose (83.2 mM) and uncompetitively by mannose (6.7 mM).     

Modification and introduction of various radioactive labels into the sialic acid moiety of sialoglycoconjugates

A method for modifying and isotopic labeling the sialyl moiety of sialoglycoproteins is described. The basis of the procedure is the reductive amination of the exocyclic aldehyde group, generated on sialic acid by mild periodate oxidation, with a variety of amino compounds and sodium cyanoborohydride. Optimal conditions were selected to obtain maximum modification of sialic acid and minimal non-specific incorporation of the amino compound (glycine). The glycine modified model glycoproteins (α₁-acid glycoprotein, fetuin) yielded single homogenous peaks upon gel filtration and on ion exchange chromatography. On gel electrophoresis a major band accounting for 92–98% of the modified glycoprotein and two minor bands consisting of dimers and trimers of the glycoprotein were observed. The modification did not alter the ability of the sialoglycoproteins to bind to wheat germ agglutinin-Sepharose or to interact with antibodies. The modified sialic acid was only partially released by mild acid hydrolysis suggesting that the introduction of an amino compound into the polyol chain of sialic acid has a stabilizing effect on the ketosidic linkage of the sugar. Interestingly, the modification rendered the sialic acid resistant to a variety of sialidases. The potential uses of this modification procedure include 1) the introduction of different isotopic labels (³H,¹⁴C,³⁵S,¹²⁵I) into the sialic acid moiety of glycoproteins; 2) the preparations of biologically active sialoglycoprotein (hormones, enzymes, co-factors) with increased circulating half-lives in animals; 3) preparation of substrates to search for endoglycosidases; 4) the direct comparison of sialoglycoprotein patterns obtained in small amounts from normal and pathological cells or tissues, and 5) the isolation and purification of cell surface sialoglycoproteins.     

Steroid-protein interactions. Fluorescence quenching of progesterone-binding globulin and alpha1-acid glycoprotein upon binding of steroids.

The influence of progesterone and four other steroids on the intrinsic fluorescence of progesterone-binding globulin was investigated. The corresponding effect of progesterone on α₁-acid glycoprotein was also studied. The intrinsic fluorescence of the progesterone-binding globulin and of α₁-acid glycoprotein was quenched by about 60 and 17%, respectively, upon forming stoichiometric complexes with progesterone. Graphical analysis of fluorescence quenching titrations with progesterone gave affinity constants at 23 °C of 2 × 10⁹m^?1 for progesterone-binding globulin and 1 × 10⁶m^?1 for α₁-acid glycoprotein. With progesterone-binding globulin, affinity constants of 1 × 10⁹m^?1 were determined for desoxycorticosterone, 1 × 10⁸m^?1 for testosterone, and 2 × 10⁶m^?1 for cortisol. The fluorescence quenching of PBG by 5-pregnen-3β-ol-20-one, 5α-pregnanedione, and 5β-pregnanedione, steroids lacking the Δ⁴-3-keto grouping, was too small to be evaluated; however, binding of the pregnanediones to progesterone-binding globulins was demonstrated when the progesterone-progesterone-binding globulin complex was “unquenched” as a result of competitive displacement of progesterone by addition of the pregnanediones. The quenching phenomenon is assumed to be mainly due to radiationless transfer from protein to the near uv (n → π1) absorption band of steroids containing the Δ⁴-3-keto chromophore.     

Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K_m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10^?4 M, 3.9·10^?4 M and 1.6·10^?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg²⁺ and Fe³⁺ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (K_i=5.6·10^?4 M) and by L-(+)-tartaric acid (K_i = 8.5·10^?5 M), while it was inhibited noncompetitively by molybdate K_i = 5.0·10^?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K_m, thermal stability and inhibition by NaF.     

Coconut α-d-galactosidase isoenzymes: isolation,purification and characterization

α-d-Galactosidases (α-d-galactoside galactohydrolase, EC 3.2.1.22) from normal coconut endosperm were isolated and partially purified by a combination of ammonium sulfate fractionation, SP-Sephadex C50–120 ion-exchange chromatography and Sephadex G-200 and G-100 gel filtration. Two molecular forms of the enzyme, designated as A and B, were eluted after SP-Sephadex C50–120 ion-exchange chromatography. α-d-Galactosidase A, which is the major isoenzyme, was partially purified 43-fold on Sephadex G-200 and has a MW of about 23 000 whereas α-d-galactosidase B was partially purified 23-fold on Sephadex G-100 and has a similar MW of about 26 600. Both isoenzymes exhibited optimum activity at pH 7.5. The apparent K_m and V_max of α-d-galactosidase A were obtained at 3.46 × 10^?4M and 1.38 × 10^?3 M p-nitrophenyl α-<d-galactoside, respectively. A distinct substrate inhibition was noted. The enzyme was inhibited strongly by d-galactose and to a lesser extent by myo-inositol, d-glucose-6-phosphate, l-arabinose, melibiose and iodoacetic acid. Similarly, makapuno α-d-galactosidase was localized in the 40–70 % (NH₄)₂SO₄ cut but its optimum activity at pH 7.5 was considerably lower as compared to the normal. Its K_m was obtained at 6.75 × 10^?4 M p-nitrophenyl α-d-galactoside while the V_max was noted at 5.28 × 10^?3 M p-nitrophenyl α-d-galactoside. Based on the above kinetic data, the possible cause(s) of the deficiency of α-d-galactosidase activity in makapuno is discussed.     

Low- and high-affinity concanavalin A binding to thymocyte plasma membrane vesicles

Binding of concanavalin A to isolated thymocyte membrane vesicles occurs through (a) numerous (～6 × 10⁶/cell equivalent) low-affinity sites (K_a = 1.3 × 10⁵ M^?1) and (b) fewer (～0.4 × 10⁶/cell equipment) specific receptors (K_a = 6.8 × 10⁶ M^?1) defined as 55,000 D glycoprotein and its multimers. Specific binding is positively-cooperative, with a Hill coefficient of～1.8. Low concentrations of glutaraldehyde selectively crosslink the 55,000 D glycoprotein with replacement of positively-cooperative sites by high-affinity sites. It is proposed that concanavalin A-binding induces multimerization of the 55,000 D glycoprotein.     

Partial purification and characterization of peroxidases from the leaves of Sapindus mukorossi

Peroxidases were isolated from Sapindus mukorossi (Reetha) and partially purified using acetone precipitation, ion-exchange chromatography with a 14-fold purification, 22% recovery and a specific activity of 266?×?10³ units/mg protein. Sapindus peroxidases (SPases) showed six bands after acetone precipitation and one distinct band after ion exchange chromatography on Native-PAGE after zymography. Enzymes purified by ion exchange chromatography were loaded on Sepahdex G-50 superfine column and their molecular weight was reported to be 25?kDa. They showed temperature optima at 50°C and pH optima at 5.0.?km for SPases was reported to be 1.05?mM and 0.186?mM for guaiacol and H₂O₂ respectively. The V_max/K_m value for o-dianisidine was 449 while for H₂O₂ it was 5?×?10⁵. Protocatechuic acid acts as a potent inhibitor for SPases (6.0% relative activity at 4.5???M) but ferulic acid inhibits its activity at a much lower concentration (0.02???M). Enzymes were stimulated by metal cations like Cu²⁺, Ca²⁺ (145, 168; percentage relative activity respectively) and showed mild inhibition (up to 20%) with Mn²⁺ and Mg²⁺. Alanine stimulated the enzyme activity (up to 33%; at 0?C100???M) while other amino acids like cysteine, methionine, tryptophan and tyrosine inhibited the SPases (13?C57% at 0?C100???M).     

Purification,characterization and gene analysis of a new α-glucosidase from <Emphasis Type="Italic">shiraia</Emphasis> sp. SUPER-H168

A new α-glucosidase from Shiraia sp. SUPER-H168 under solid-state fermentation was purified by alcohol precipitation and anion-exchange and by gel filtration chromatography. The optimum pH and temperature of the purified α-glucosidase were 4.5 and 60 °C, respectively, using p-nitrophenyl-α-glucopyranoside (α-pNPG) as a substrate. Ten millimoles of sodium dodecyl sulfate, Fe²⁺, Cu²⁺, and Ag⁺ reduced the enzyme activity to 0.7, 7.6, 26.0, and 6.2 %, respectively, of that of the untreated enzyme. The K _m, V _max, and k _cat/K _m of the α-glucosidase were 0.52 mM, 3.76 U mg^?1, and 1.3?×?10⁴ L s^?1 mol^?1, respectively. K _m with maltose was 0.62 mM. Transglycosylation activities were observed with maltose and sucrose as substrates, while there was no transglycosylation with trehalose. DNA and its corresponding full-length cDNA were cloned and analyzed. The α-glucosidase coding region consisted of a 2997-bp open reading frame encoding a 998-amino acid protein with a 22-amino acid signal peptide; one 48-bp intron was located. The α-glucosidase was a monomeric protein with a predicted molecular mass of 108.2 kDa and a predicted isoelectric point of 5.08. A neighbor-joining phylogenetic tree demonstrated that Shiraia sp. SUPER-H168 α-glucosidase is an ascomycetes α-glucosidase. This is the first report of α-glucosidase from a filamentous fungus that had good glycoside hydrolysis with maltose and α-pNPG, transglycosylation and conversion activity of maltose into trehalose.     

Purification and characterization of a class II α-Mannosidase from Moringa oleifera seed kernels

α-Mannosidase (EC. 3.2.1.114) belonging to class II glycosyl hydrolase family 38 was purified from Moringa oleifera seeds to apparent homogeneity by conventional protein purification methods followed by affinity chromatography on Con A Sepharose and size exclusion chromatography. The purified enzyme is a glycoprotein with 9.3 % carbohydrate and exhibited a native molecular mass of 240 kDa, comprising two heterogeneous subunits with molecular masses of 66 kDa (α-larger subunit) and 55 kDa (β-smaller subunit). Among both the subunits only larger subunit stained for carbohydrate with periodic acid Schiff’s staining. The optimum temperature and pH for purified enzyme was 50 °C and pH 5.0, respectively. The enzyme was stable within the pH range of 3.0–7.0. The enzyme was inhibited by EDTA, Hg²⁺, Ag²⁺, and Cu²⁺. The activity lost by EDTA was completely regained by addition of Zn²⁺. The purified enzyme was characterized in terms of the kinetic parameters K _m (1.6 mM) and V _max (2.2 U/mg) using para-nitrophenyl-α-D-mannopyranoside as substrate. The enzyme was very strongly inhibited by swainsonine (SW) at 1 μM concentration a class II α-Mannosidase inhibitor, but not by deoxymannojirimycin (DMNJ). Chemical modification studies revealed involvement of tryptophan at active site. The inhibition by SW and requirement of the Zn²⁺ as a metal ion suggested that the enzyme belongs to class II α-Mannosidase.     

Analysis of lidocaine interactions with serum proteins using high-performance affinity chromatography

High-performance affinity chromatography was used to study binding by the drug lidocaine to human serum albumin (HSA) and α₁-acid glycoprotein (AGP). AGP had strong binding to lidocaine, with an association equilibrium constant (K_a) of 1.1–1.7 × 10⁵ M^?1 at 37 °C and pH 7.4. Lidocaine had weak to moderate binding to HSA, with a K_a in the range of 10³ to 10⁴ M^?1. Competitive experiments with site selective probes showed that lidocaine was interacting with Sudlow site II of HSA and the propranolol site of AGP. These results agree with previous observations in the literature and provide a better quantitative understanding of how lidocaine binds to these serum proteins and is transported in the circulation. This study also demonstrates how HPAC can be used to examine the binding of a drug with multiple serum proteins and provide detailed information on the interaction sites and equilibrium constants that are involved in such processes.     

Cordil Reversibly Inhibits the Na,K-ATPase from Outside of the Cell Membrane. Role of K-dependent Dephosphorylation

Abstract

Cordil-LND796 is a new cardiotonic glycoside under development. In rat brain microsomes where three isoforms of the Na, K-ATPase with differential affinities for cardiac glycosides have been identified, Cordil had higher affinity for the α₃ (IC₅₀ = 0.02 μM) than for the α₂ (IC₅₀ = 0.6 μM) and the α₁ (IC₅₀ = 30 μM) isozymes. Cordil is potentially a selective inhibitor for both α₂ and α₃ Na, K-ATPase isoforms. Using inside out vesicles we have shown that Cordil binds to and inhibits Na, K-ATPase at an extracellular site. The dissociation kinetic rates (k_?1) from the ATPase and the phosphatase activity (K-dependent dephosphorylation) of the Na, K-ATPase were similar for Cordil. Despite these similarities to ouabain comparison of the kinetics of the Na, K-ATPase inhibition by ouabain and Cordil revealed marked differences in their association rates (k₊₁ = 0.7 1 mol¹ min^?1 and k₊₁ = 6 × 10_?3 1 mol_?1 min_?1 respectively) and their dissociation rates (k_?1 = 1.3 ± 0.2 × 10^?4 S^?1 and k_?1 = 69 ± 7 × 10^?4 s^?1 respectively). Both binding association and dissociation rates were enhanced for Cordil. These data are compatible with a stabilizing effect of Cordil on the E₂P conformational state of Na, K-ATPase.     

A Review of: “Biochemistry and Methodology of Lipids,Eds. A.R. Johnson and J.B. Davenport Wiley-Interscience,New York, 1971 x + 578 pages,ill., hard cover; $29.50”

A previous communication from this laboratory¹ as well as one from another³ described the separation of α₂-macroglobulin from swine serum. The products from both laboratories contained, in addition to α₂-macroglob-ulin, an additional macroglobulin contaminant with α₂-globulln mobility. Due to their physicochemical similarity these macroglobulins are not resolved using conventional column procedures such as ion exchange chromatography and gel filtration. Subsequent experiments have shown that immunoelectro-phoretically pure swine α₂-macroglobulln is present, in good yield (65%) in the breakthrough effluent of columns of Bio-Gel A-1.5m-Reactive Blue 2 while the contaminating macroglobulin is tightly bound. The production of highly purified swine α₂-macroglobulin utilizing this observation is the subject of the present report. The product of the separation was found to be homogeneous when subjected to Immunoelectrophoresis, at a concentration of 14–16 mg/ml, and diffused against antiswlne whole serum antibody. The production of monospecific antibody, a more stringent test for homogeneity, resulted when the purified α₂-macroglobulin was injected into rabbits. Physicochemical analyses on the purified product showed that swine and human α₂-macro-globulins are true homologs.     

Purification and partial characterization of the extradiol dioxygenase, 2′-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase,in the fluorene degradation pathway from Rhodococcus sp. strain DFA3

Type II extradiol dioxygenase, 2′-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase (FlnD1D2) involved in the fluorene degradation pathway of Rhodococcus sp. DFA3 was purified to homogeneity from a heterologously expressing Escherichia coli. Gel filtration chromatography and SDS-PAGE suggested that FlnD1D2 is an α₄β₄ heterooctamer and that the molecular masses of these subunits are 30 and 9.9 kDa, respectively. The optimum pH and temperature for enzyme activity were 8.0 and 30 °C, respectively. Assessment of metal ion effects suggested that exogenously supplied Fe²⁺ increases enzyme activity 3.2-fold. FlnD1D2 catalyzed meta-cleavage of 2′-carboxy-2,3-dihydroxybiphenyl homologous compounds, but not single-ring catecholic compounds. The K_m and k_cat/K_m values of FlnD1D2 for 2,3-dihidroxybiphenyl were 97.2 μM and 1.5 × 10^?2 μM^?1sec^?1, and for 2,2′,3-trihydroxybiphenyl, they were 168.0 μM and 0.5 × 10^?2 μM^?1sec^?1, respectively. A phylogenetic tree of the large and small subunits of type II extradiol dioxygenases suggested that FlnD1D2 constitutes a novel subgroup among heterooligomeric type II extradiol dioxygenases.     

A novel xylan degrading β-d-xylosidase: purification and biochemical characterization

Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular ??-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. ??-xylosidase is a glycoprotein (39?% carbohydrate content) and has a molecular mass of 137?kDa by SDS-PAGE, with optimal temperature and pH at 70?°C and 3.0?C5.5, respectively. ??-xylosidase was stable in acidic pH (3.0?C6.0) and 70?°C for 1?h. The enzyme was activated by 5?mM MnCl₂ (28?%) and MgCl₂ (20?%) salts. The ??-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-??-d-xylopyranoside, exhibiting apparent K_m and V_max values of 0.66?mM and 39?U (mg protein)^?1 respectively, and to a lesser extent p-nitrophenyl-??-d-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel ??-d-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by ??-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.     

Interactions of cyclic hexapeptide cyclo(Pro-Sar-Sar)2 with small molecules

N.m.r. and c.d. spectroscopy have been used to study the interactions of cyclic hexapeptide cyclo(Pro-Sar-Sar)₂ with metal ions and ammonium ions. Cyclo(Pro-Sar-Sar)₂ was found to form complexes with Li⁺, K^?, Ba²⁺ and Cu²⁺, accompanying the conformational change into a single conformer, and the conformation of cyclo(Pro-Sar-Sar)₂ in the Li⁺-complex was different from that in the Cu²⁺-complex. These findings indicate conformational flexibility of cyclo(Pro-Sar-Sar)₂. The equilibrium constant for the complexation with Li⁺ was 2.3 × 10²l mol^?1, and cyclo(Pro-Sar-Sar)₂ adopted an asymmetric conformation in the complex. The addition of α-amino acid ester hydrochloride also caused the conformational change of cyclo(Pro-Sar-Sar)₂), but in this case it did not converge into a single conformation. This type of interaction was strengthened with aromatic α-amino acid ester hydrochloride due to the aromatic-amide interactions. Finally, the rates of exchange between unbound α-amino acid ester hydrochlorides and those complexed with cyclo(Pro-Sar-Sar)₂ were found to be different, according to the nature of α-amino acid.     

Identification of Protease from Euphorbia amygdaloides Latex and its Use in Cheese Production

Abstract

In this research, protease enzyme was purified and characterized from milk of Euphorbia amygdaloides. (NH₄)₂SO₄ fractionation and CM‐cellulose ion exchange chromatography methods were used for purification of the enzyme. The optimum pH value was determined to be 5, and the optimum temperature was determined to be 60°C. The V_max and K_M values at optimum pH and 25°C were calculated by means of Linewearver‐Burk graphs as 0.27 mg/L min^?1 and 16 mM, respectively. The purification degree was controlled by using SDS‐PAGE and molecular weight was found to be 26 kD. The molecular weight of the enzyme was determined as 54 kD by gel filtration chromatography. These results show that the enzyme has two subunits.

In the study, it was also researched whether purified and characterized protease can be collapsed to milk. It was determined that protease enzyme can collapse milk and it can be used to produce cheese.     

α-Ketoglutaric acid production from rapeseed oil by Yarrowia lipolytica yeast

The possibility of using rapeseed oil as a carbon source for microbiological production of α-ketoglutaric acid (KGA) has been studied. Acid formation on the selective media has been tested in 26 strains of Yarrowia lipolytica yeast, and the strain Y. lipolytica VKM Y-2412 was selected as a prospective producer of KGA from rapeseed oil. KGA production by the selected strain was studied in dependence on thiamine concentration, medium pH, temperature, aeration, and concentration of oil. Under optimal conditions (thiamine concentration of 0.063 μg?g cells^?1, pH?3.5, 30 °C, high dissolved oxygen concentration (pO₂) of 50 % (of air saturation), and oil concentration in a range from 20 to 60 g?l^?1), Y. lipolytica VKM Y-2412 produced up to 102.5 g?l^?1 of KGA with the mass yield coefficient of 0.95 g?g^?1 and the volumetric KGA productivity (Q _KGA) of 0.8 g?l^?1?h^?1.     

Solution properties and chain flexibility of pullulan in aqueous solution

Molecular characteristics for pullulan, a polysaccharide produced by a fungus Aureobasidium pullulans, were measured by light scattering, viscometry, and gel-permeation chromatography. From the experimental data the Mark-Houwink-Sakurada viscosity equation in water at 25°C was determined for samples having the molecular weight M ranging from 48 × 10³ to 2.18 × 10⁶ g mol^?1 as [η] = (1.91 ± 0.02) × 10^?2M_w^0.67±0.01 (in cm³ g^?1); and as molecular weight decreased, the slope of the viscosity equation decreased, although the molecular weight values below 30 × 10³ g mol^?1 evaluated by gel-permeation chromatography were somewhat unreliable. The unperturbed dimensions 〈R²〉₀^1/2 of pullulan were estimated by determining the expansion factor α_s, from the theoretical combination of theories for the interpenetration function Ψ and those for α_s. The 〈R²〉₀/M value estimated from this procedure in 6.7 × 10^?17 cm² mol g^?1. We concluded that the polysaccharide chain that is linked by the α-1,6-glucosidic linkage behaves like a flexible chain in aqueous solution.