A sensitive method for estimating the carbohydrate content of glycoproteins

A spectrophotometric method is described for estimating the carbohydrate content of acid-hydrolyzed glycoproteins. The method is based on the alkaline ferricyanide reaction with reducing sugars and is accurate in the 1–25 nmole range. Using a simple two-step ion-exchange column procedure both the total sugar content and the proportion of neutral and amino sugars can be determined.     

A simple and reliable method for the detection of sialylation in complex/hybrid-type carbohydrate chains of glycoproteins by mixed lectins

A practical and convenient method for discriminating between the presence and the absence of sialic acid in carbohydrate chains of glycoproteins was devised using paramagnetic beads and two lectins, Sambucus sieboldiana lectin (SSA) and Ricinus communis agglutinin (RCA120). The glycoproteins of transferrin or thyroglobulin were firstly captured to paramagnetic beads that were previously coated with corresponding antibody, and then the lectins of RCA120-biotin and SSA-FITC were bound to the glycoproteins on the paramagnetic beads. Finally, the fluorescence intensity of the beads was measured to determine the ratios of lectins RCA120-biotin/Cy5-streptavidin to SSA-FITC. The mixed lectins method showed bigger difference of the ratios between the presence and the absence of sialic acid, indicating higher discrimination efficiency than the ordinary non-mixed lectins method. Furthermore, statistical analysis by two-side t-test indicated that the mixed lectins method was more highly reliable than the ordinary non-mixed lectins method in discriminating between the presence and the absence of sialic acid. The reaction with the two lectins can be performed in a single tube and readily automated taking advantage of the use of paramagnetic beads.     

The biosynthesis of carbohydrate chains of glycoproteins and their heterogeneity

Current state of research on the mechanism of biosynthesis of carbohydrate chains of N- and O-glycoproteins is reviewed. Functional predetermination of a multistage mechanism of the carbohydrate components' biosynthesis in N-glycosylproteins is suggested. Origin and character of heterogeneity of the carbohydrate chains in these biopolymers are discussed.     

Composition and distribution of carbohydrate chains in glycoproteins of human erythrocyte membrane

The M-, N-, and MN-glycoproteins obtained from human erythrocytes by phenol-water extraction were purified by gel filtration and digested with Pronase and trypsin. The products of degradation were fractionated by gel filtration on Sephadex G-25 and DEAE-Sephadex A-50 and the fractions were examined by poly(acrylamide)-gel electrophoresis in the presence of dodecyl sodium sulfate, analyzed for carbohydrate and amino acid contents, and tested for M and N blood-group activity. From the results, it is suggested that the glycoprotein chains are composed of a hydrophobic moiety devoid of carbohydrate chains and a hydrophilic moiety containing carbohydrate chains of different compositions, irregularly distributed along the protein chains and linked to L-asparagine, L-serine, or L-threonine residues. The M and N activity typical for the undegraded glycoproteins, and the “basic” or “precursor-type” N activity, were found in different glycopeptide fractions.     

Structures of the carbohydrate chains of membrane glycoproteins IIb and IIIa of human platelets

Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.     

A rapid and sensitive method for the analysis of carbohydrate components in glycoproteins using gas-liquid chromatography

A rapid and simple gas-liquid chromatographic method for the determination of subnanomolar amounts of carbohydrates derived from glycoproteins is described. The procedure involves methanolysis in the presence of methyl acetate followed by removal of hydrogen chloride by coevaporation with t-butyl alcohol and trimethylsilylation. The method is also applicable to samples containing uronic acids and lipids.     

A new method for the isolation of renal basement membranes

A method is described for the isolation of basement membranes from rabbit renal cortex in which the detergent $\text{N-}\text{lauroyl}$ sarcosine is used as the disruptive agent. The isolated membranes have been compared with membranes prepared using ultrasonication and they were comparable both in terms of purity and gross chemical composition. Glomerular and tubular basement membranes were isolated by first separating glomeruli from tubules by density gradient centrifugation followed by detergent treatment of the separated tissues.The detergent method has the advantage that the basement membranes retained their native structure to a large degree, whereas sonicated membranes were severely fragmented. Collagen fibres were a significant contaminant in both preparations and were revealed more clearly by negative staining than by examination of thin sections. Studies with the detergent-treated membrane revealed that a few proteins, which seemed to be membrane components, were extracted with 1 M NaCl and that these proteins were lost from the basement membranes during sonication used in the conventional isolation procedure.     

A new method for the selective isolation of phosphoserine-containing peptides

Meyer et al. [(1986) FEBS Lett 204, 61-66] have shown that phosphoserine can be converted to S-ethylcysteine by beta-elimination and addition of ethanethiol. I have utilised this modification to develop a rapid method for the selective purification of phosphoserine-containing peptides from complex mixtures. Changing phosphoserine to S-ethylcysteine increases the hydrophobicity of a peptide, altering its mobility during reverse-phase chromatography. The number of S-ethylcysteine residues in a peptide can be quantified at the picomolar level, following acid hydrolysis and conversion to the phenylthiocarbamyl derivative. The procedure may be particularly powerful for the analysis of peptides that are phosphorylated at multiple sites in vivo.     

A new isolation and purification method for staphylococcal protein A using membrane encapsulated rabbit IgG-agarose

A new isolation and purification method for bioproducts using membrane-encapsulated affinity adsorbents was investigated. The new method involves encapsulation of affinity adsorbents, batch adsorption of the bioproduct from whole fermentation broth and rapid batch desorption after dissolution of the capsule membranes. Recovery of protein A from Staphylococcus aureus was used as the model experimental system. Affinity adsorbents such as rabbit IgG-agarose were successfully encapsulated within calcium alginate membranes and used directly to recover protein A from whole cell homogenate containing a number of macromolecular contaminants as well as suspended solids. Both high yield and high purity of protein A were recovered by this method in comparison with various previously reported methods.