Periodate oxidation studies of human pituitary follicle-stimulating hormone

A preparation of human pituitary follicle-stimulating hormone was subjected to periodate oxidation, borohydride reduction and acid hydrolysis. Comparison of the analysis of the remaining intact carbohydrate and amino acid units with the analyses of the original material and identification of the carbohydrate fragments permit some structural assignments to the molecule of follicle-stimulating hormone. The results of radioimmunological assay of fragments of the molecule of follicle-stimulating hormone suggest that, although the carbohydrate component is essential for biological activity, it is not a requirement for immunological activity, which appears to be a function of the protein moiety.     

Periodate oxidation and degradation studies on the major water-soluble arabinoxylan in rye grain

The major water-soluble arabinoxylan from rye grain has previously been shown to contain a main chain of 4-linked β- -xylopyranosyl residues in which, on average, every second is substituted at position 3 with terminal - -arabinofuranosyl residues. Periodate oxidation, reduction and fragmentation by mild acid hydrolysis produced a series of glycerol xylosides containing 4-linked xylopyranosyl residues linked at the reducing end to position 2 of glycerol. It was shown that a one-step periodate oxidation was incomplete due to the formation of relatively stable hemiacetal linkages. A sequential oxidation and reduction procedure was used to bring about complete oxidation of arabinose and unbranched xylose residues in the intact polysaccharide. Quantitative analysis of the products liberated by mild acid hydrolysis revealed the presence of glycerol xylosides with one, two or three xylose residues in the molar ratio of 1·00:0·86:0·02. The xylose residues must have originated from branched residues in the main chain of the arabinoxylan. The units or small blocks of two residues are therefore distributed mainly as isolated branched residues and not randomly as previously reported.     

Visual detection of carcinoembryonic antigen on surfaces.

An attempt is made to detect carcinoembryonic antigen (CEA) at clinically interesting concentrations by using a simple immunologic surface test. Antibodies to CEA are detected in a direct test at concentrations below 1 ng/ml. The sensitivity of this assay is mainly limited by diffusion to the reacting surface. CEA is detected in an inhibition test at concentrations down to 20 ng/ml. The sensitivity of this inhibition test is limited by the average equilibrium constant in solution of the antibody-antigen reaction.     

Periodate oxidation of methionine in proteins

Treatment of free methionine with an equimolar amount of periodate gave nearly quantitative formation of the sulfoxide; treatment of free methionine sulfoxide with equimolar periodate gave nearly equal amounts of the original sulfoxide and the sulfone. Treatments of 0.5-1.0% solutions of the following proteins with relatively low concentration of periodate (5 mm) gave the following approximate values for conversion of methionine sulfoxide from total methionine: bovine pancreatic ribonuclease A (2 of 4), chicken ovalbumin (14 or 17), chicken ovotransferrin (5 of 11), human serum transferrin (2 of 8), bovine α-chymotrypsin (1 of 2). It is recommended that when proteins are treated with sodium periodate (and probably with oxidizing agents in general), especially when changes in properties are observed, determinations of methionine sulfoxide should be done.     

Periodate oxidation and the shapes of glycosaminoglycuronans in solution.

1. It is proposed that periodate oxidation of glycol groups in the repeating units of polysaccharide molecules can be used to probe differences in polymer shapes in solution. 2. Measurement of second-order rate constants (k2) of periodate-glycol reactions may be compared between polymers and relevant monomers, to assess perturbations due to polymer configuration. 3. Factors effecting the measurement and interpretation of k2 are discussed. Over-oxidation, free-radical side reactions, end-group effects, Donnan equilibria and polymer (or molecular-weight) effects are relevant, but their importance is either small or can be minimized in practice. 4. A small group of glycosaminoglycuronans (chondroitin 4- and 6-sulphates and hyaluronate) are oxidized 50--100 times more slowly than three other glycosaminoglycuronans of similar composition, relevant monomers or three homopolyuronides. 5. A stable configuration in solution is postulated for the periodate-resistant polymers, involving carboxylate, acetamido and hydroxy groups in hydrogen-bonded sequences on alternate sides of the molecule. The more easily oxidizable polyuronides are unable to form this configuration. 6. The effect of temperature on the postulated configuration is investigated through the Arrhenius plot of k2, measured to hyaluronate, chondroitin 6-sulphate and methyl 4-O-methyl-alpha-D-glucopyranoside. Probable transitions at high (around 90 degrees C) temperatures were observed for both polymers, with an additional transition at about 37 degrees C in the case of hyaluronate. 7. L-Iduronic acid can take up different conformations depending on the polymer environment.     

Periodate oxidation studies in the elucidation of the structures of sialic acid containing oligosaccharides

The structures of sialo-oligosaccharide alditols as determined by ¹H-NMR spectrometry together with methylation analysis did not correspond with those derived previously from quantitative periodate oxidation data alone. Possible causes of the discrepancy were explored in the periodate oxidation methodology. No free sialic acid was released by the acidity of the periodate in the course of oxidation at pH 4.5. The anionic properties of the sialic acid residues were therefore utilized to separate the periodate oxidation products and thereby establish the position of the sialic acid in the oligosaccharide chain.     

Structural studies on the immunogenic form of the enterobacterial common antigen.

It has been shown that enterobacterial common antigen is chemically linked to the hexose region of the R1-type lipopolysaccharide fo the Escherichia coli strain F470 which is immunogenic for this antigen. The number of R core stubs substituted is very small but it is a-parently sufficient to induce antibody formation to the enterobacterial common antigen in the rabbit.     

Biosynthesis and glycosylation of the carcinoembryonic antigen.

Human gastric adenocarcinoma MKN-45 cells were found to synthesize actively carcinoembryonic antigen (CEA). The biosynthesis and carbohydrate processing of CEA were studied in these cells by means of metabolic labelling followed by immunoadsorption with a specific polyclonal-antibody preparation and gel electrophoresis. Pulse-chase studies with [14C]leucine and [3H]mannose (shortest pulse 3 min) showed that N-linked oligosaccharide side chains are added to the protein co-translationally, producing a high-mannose immature CEA; the average molecular mass of this form is 145 kDa. The protein is later translocated to the Golgi apparatus and here undergoes additional processing; these modifications are visible in our system as a broadening of the CEA band and require about 4 h. The upper limit of mature CEA band reaches 200 kDa, but radioactivity is maximally incorporated at 168 kDa. The extent of co-translational glycosylation was measured by treating the cells with tunicamycin; in the presence of this inhibitor, a 74 kDa aglyco-CEA was produced and was still recognized by the antibody. Monensin, an ionophore which interferes with glycoprotein maturation and terminal sugar addition, blocked broadening of the CEA band, producing a sharp 141 kDa peak. In conclusion, CEA appears to be synthesized as a 145 kDa high-mannose immature form, the protein core accounting for about half of its molecular mass. Full maturation results in a broad band at 168 kDa.     

Immunohistochemical studies of colorectal carcinoma with a monoclonal antibody against carcinoembryonic antigen

Immunoperoxidase localization of carcinoembryonic antigen (CEA) was performed on tissue sections of colorectal carcinoma using a monoclonal antibody (MAb) against CEA. CEA has been demonstrated in 20 out of 22 rectum carcinomas (90.9%), in all of 23 colonic carcinomas, in none of 4 hyperplastic polyps and in 2 out of 6 adenomatous polyps (33.3%). CEA was found more often, and the intensity of the staining was stronger in well-differentiated carcinomas than in moderately and poorly differentiated carcinomas. No correlation was found between the presence of CEA in colorectal carcinoma and the stages of the disease. The mean values of serum CEA in patients with colorectal carcinoma and polyps with negative, weakly and strongly positive staining were 5.4 +/- 3.9 ng/ml, 28.3 +/- 23.8 ng/ml and 99.8 +/- 145.3 ng/ml respectively. Elevation of serum CEA occurred in 30 out of 39 (78.9%) cases with strongly positive CEA staining, in 4 out of 6 (66.7%) with weakly positive and in 1 out 9 (11.1%) with negative staining. A significant difference was found in serum CEA activity between the group with negative CEA staining and positive CEA staining (P less than 0.01). Our results suggest that the monoclonal antibody (MAb C27) can be used for the localization of CEA in conventionally prepared tissues of colorectal carcinomas by immunoperoxidase techniques for routine immunopathological diagnosis.     

Periodate oxidation and alkaline degradation of heparin-related glycans

Heparin, heparan sulphate, and various derivatives thereof have been oxidised with periodate at pH 3.0 and 4° and at pH 7.0 and 37°. Whereas oxidation under the latter conditions destroys all of the nonsulphated uronic acids, treatment with periodate at low pH and temperature causes selective oxidation of uronic acid residues. The reactivity of uronic acid residues depends on the nature of neighbouring 2-amino-2-deoxyglucose residues. d-Glucuronic acid residues are susceptible to oxidation when flanked by N-acetylated amino sugars, but resistant when adjacent residues are either unsubstituted or N-sulphated. L-Iduronic acid residues in their natural environment (2-deoxy-2-sulphoamino-d-glucose) are resistant to oxidation, whereas removal of N-sulphate groups renders a portion of these residues periodate-sensitive. Oxidised uronic acid residues in heparin-related glycans may be cleaved by alkali, producing a series of oligosaccharide fragments. Thus, periodate oxidation-alkaline elimination provides an additional method for the controlled degradation of heparin.     

Isolation of carcinoembryonic antigen]

A method employed in the authors' laboratory for isolating the carcinoembryonic antigen (CEA) from tumours of the entodermal digestive tract and their metastases is described. The main step is fractionation of the preparations obtained by perchloric acid extraction using gel filtration on Sephadex G-200, Sepharose 4 B, and Sepharose 6 B at pH 4.5 and 7.0. The peaks with CEA-specific antigeneity were characterized by the distribution coefficient of the CEA between the mobile and the stationary phases (Kav), and by the relative elution volume (ve/vo). The CEA purified by gel filtration could not reliably be enriched through preparative electrophoresis in Sephadex G-25 gel, polyacrylamide gel or Pevikon powder. The isolation procedures used were compared with those reported in the literature, and the results obtained are discussed.     

Periodate oxidation of chitosans with different chemical compositions

Periodate oxidation of chitosans with different chemical compositions were investigated by determining the consumption of periodate consumed, and the amount of ammonia and formaldehyde liberated during the reaction. Oxidised chitosans were further characterised by size-exclusion chromatography with online multi-angle light scattering (SEC-MALLS) to obtain the molecular weight distributions, and by elemental analysis to obtain the N/C ratio. Chitosans became only partially oxidised by periodate, reaching degrees of oxidation around 0.5, when oxidising with excess periodate. Overconsumption of periodate is attributed to the extensive depolymerisation, which occurs concomitantly with the oxidation, thereby exposing novel reducing and non-reducing ends which consume additional periodate. Both the rate and extent of overoxidation, and the rate of depolymerisation decreased with increasing F(A). A chitosan-specific degradation mechanism is probably involved in the depolymerisation in addition to the general free-radical-mediated degradation.     

Relative positions of some epitopes on carcinoembryonic antigen

Summary To investigate whether anti-(carcinoembryonic antigen) monoclonal antibodies (mAb) react with single or repeated epitopes, sandwich radioimmunoassays in homologous and heterologous combinations were performed. Four mAb (I-27, I-47, II-17 and to some degree II-16) gave homologous binding while two mAb (I-38S1 and II-10) did not. Taken together with previous immunoprecipitation studies we conclude that all these mAb except II-10 react with repeated epitopes. The relative positions of the epitopes recognized by these mAb and of three additional mAb (II-6, II-7 and CB-CEA-1) were investigated using a plate antibody competition test with enzyme-labelled carcinoembryonic antigen (CEA). mAb I-38S1, II-6, II-7, II-10, II-16 and CB-CEA-1 were mutually cross-reactive, and were classified as belonging to one epitope group. mAb I-27 and I-47 fell outside this group and did not interfere with the binding of CEA conjugate to mAb II-17 either. They therefore represent a second epitope group. mAb II-17 showed no interference with the binding of CEA to any of the other mAb and must therefore represent a third epitope group. The slopes of the plate antibody competition curves were used for calculation of a correlation matrix, which in turn was used to depict the relative positions of the epitopes recognized by the mAb in the large group.