UBC Theses and Dissertations
Development of methods for the simultaneous visualization of neutral sugars and either sialic acid and its side chain O-acyl variants or O-sulphate ester based on the selective periodate oxidation of sialic acid Volz, Doris Elenore
The objective of this study was to establish conditions for the selective periodate oxidation of sialic acid, and then use these conditions to develop a series of general methods for the simultaneous visualization of "neutral sugars" (ie. hexose, 6-deoxyhexose and N-acetylhexosamine) and sialic acid and its side chain O-acyl substituted variants, or O-sulphate ester. Investigations of selective conditions for the oxidation of sialic acids demonstrated that oxidation for one hour at 4°C with 0.4 mM periodic acid in approximately 1M hydrochloric acid (PA*) oxidized all available sialic acid residues of both the sialo and sialosulphoglycoproteins of human and rat colon and the sialoglycoproteins of rat sublingual gland. These conditions produced no visible Schiff staining of either neutral macromolecules or vicinal diols located on the "neutral sugars" of sialo and sialosulphoglycoproteins, and did not result in the extraction of epithelial glycoproteins or in the de-O-acylation of side chain substituted sialic acid residues. Therefore, PA* can be used as a specific reagent for the selective oxidation of sialic acids. Studies of the mechanism of oxidation with PA* showed that the lack of PAS reactivity of "neutral sugars" was not due to the production of Schiff unreactive hemiacetals or hemialdals. It is possible that the selective oxidation of sialic acids with PA* results from an increase in the oxidation rate of sialic acid residues together with a decrease in the oxidation rate of "neutral sugars". Based upon this method for the selective oxidation of sialic acid residues (PA*), five new methods have been devised for the simultaneous visualization of "neutral sugars" and either sialic acid and it side chain 0-acyl derivatives or O-sulphate ester. The first of these is the selective periodate oxidatlon-borohydride reduction-saponification-selective periodate oxidation-thionin Schiff-saponification-borohydride reduction-periodic acid-Schiff (PA*/Bh/KOH/PA*/T/KOH/Bh/PAS) technique, in which sialic acids with O-acyl substituents at C7, C8, or C9 (or which have two or three side chain O-acyl substituents) stain blue while "neutral sugars" with periodate sensitive vicinal diols stain magenta. In the second method, the saponification-selective periodate oxidation-thionin Schiff-saponification- borohydride reduction-periodic acid-Schiff (KOH/PA*/T/KOH/Bh/PAS) method all sialic acids stain blue while "neutral sugars" stain magenta. In the third method, the selective periodate oxidation-thionin Schiff-borohydride reduction-periodic acid-Schiff-saponification (PA*/T/Bh/PAS/KOH) method, sialic acids without side chain substituents or which have an O-acyl substituent at C7 stain blue while "neutral sugars" stain magenta. In the fourth method, the saponification-selectlve periodate oxidation-borohydride reduction-alcian blue PH 1.0-periodic acid-Schiff (K0H/PA*/Bh/AB1.O/PAS) technique, O-sulphate esters stain aquamarine blue while "neutral sugars" stain magenta. In all of these techniques, mixtures of the components stain in various shades of purple. In the fifth and final method, the saponification-selective periodate oxidation-borohydride reduction-periodic acid-Schiff (KOH/PA*/Bh/PAS) technique, selective identification of "neutral sugars" in macromolecules which also contain sialic acids can be achieved.
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