Amino carbohydrate interference

Periodate followed by Schiff reagent has been successfully used for staining carbohydrate structures on histological slides,188 189 but the possibility of interference by amino acids has been pointed out.190... 

This procedure is often used when the extraction solvent chosen has been water alone, since in this case polar compounds such as salts and amino acids may contaminate the extract. Precipitation with an appropriate solvent, e.g., a mixture of acetate buffer/acetonitrile (22), is then required. Water-soluble macromolecules also interfere and may be removed to a considerable extent by simple precipitation with an organic solvent, for example, ethanol. Nevertheless this may be troublesome, since a proportion of the carbohydrates of interest may become absorbed to the precipitated material, which will be subsequently lost. On the other hand, the sugars must be sufficiently soluble in the solvent used to avoid its precipitation from the solution. 

An interesting correlation between tripeptide sequences and genetic mutations has been made by compilation of the number of occurrences of the tripeptide sequence Asn—B—Ser and Asn—B—Thr in protein sequence-data at present available from the literature.134 A total of 18,251 tripeptide sequences from 264 proteins was grouped by computer into the 400 possible tripeptide combinations of the 20 amino acids in which the second position was ignored. The total number of Asn—B—(Ser/Thr) sequences actually present in these proteins was 61, whereas the total number theoretically expected, on the basis of a random distribution within the sequence, would have been 102. The observed data were thus lower than expected by 4 standard deviations. On the other hand, such chemically similar tripeptides as Gin—B—(Ser/Thr), Asp—B—(Ser/Thr), and (Ser/Thr)—B—Asn occurred the number of times expected for random distribution. It was suggested that this low frequency of incidence of the Asn—B—(Ser/Thr) sequence results from a restriction of its occurrence in proteins by a process of natural selection. Any protein that acquired this tripeptide as a result of a mutation would be soon rejected, because carbohydrate would be bound to the asparagine residue and its presence would interfere with the normal metabolic function of the protein. 

Covalent, orientated Amino groups, hydrazine Binds to carbohydrate residues of EC region of antibodies High densihes are available, strong protein attachment, oriented immobilization/ surface interference... 

UV and fluorescent spectroscopy can be employed down to 190 nm because there is no solvent interference. Mass spectrometry is easy because the water provides good ionization. Flame ionization detection (FID) is of particular interest because potentially it offers a sensitive and universal detector. A number of different interfaces have been used, including heated capillaries, which have been examined by Miller and Hawthorne [62], Ingelse et al. [63], and others [64, 65], who separated a range of analytes including alcohols, amino acids, and phenols. An alternative method employing a cold nebuliza-tion of the eluent has been introduced by Bone et al. [66]. They were able to detect both aliphatic and aromatic alcohols, polymers, carbohydrates, parabens, and steroids. 

If free amino acids are to be analyzed in foods or feeds, it is advisable to remove matrix components such as lipids, carbohydrates, and nucleic acids from the sample. Of course, the necessary extraction steps are time-consuming, but higher concentrations of such compounds may severely interfere with the amino acid analysis. After hydrolysis of protein-free mixtures of nucleosides, nucleotides, and nucleic acids, Paddock et al. [38] even observed the formation of amino acids, predominantly glycine. 

Studies on the incorporation of labeled amino acids in thyroid slices have provided a more detailed description of the mechanism of synthesis of the hormone. The radioactivity first appears in soluble polypeptides with sedimentation coefficients of 3, 8, or 12. Puromy-cin or actinomycin blocks the incorporation of the precursor into the soluble polypeptides. The half-life of the messenger RNA for thyroglobulin polypeptide was estimated to be 15-20 hours. Indeed, after inclusion of actinomycin in the incubation mixture, thyroglobulin synthesis continues for several hours. The subunits are transferred from the site of synthesis to an assembly center, in which the subunits are iodinated, carbohydrate units are included in the molecule, and subunits are condensed into a finished protein. Puromycin fails to interfere with the formation of 19 S units. 

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