Periodate Oxidation Techniques

Enzymes that contain carbohydrate, such as HRP or GO, may be oxidized with periodate to create active derivatives that subsequently can be used to label antibodies or other targeting molecules at their amine functional groups. The aldehyde—HRP intermediate may be stored for extended periods in a frozen or lyophilized state without loss of activity (either enzymatic or coupling potential). Avoid, however, storage in a liquid state, since polymerization may occur—resulting in precipitation and loss of activity. 

The protocol for periodate oxidation of HRP and its conjugation with other proteins may be found in Chapter 10, Section 1.3. 

Molecules containing polysaccharide chains may be oxidized to possess reactive aldehyde residues by treatment with sodium periodate. Any adjacent carbon atoms containing hydroxyl groups will be affected, cleaving the carbon-carbon bond and transforming the hydroxyls into 

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Antibodies to nucleosides usually react with nucleosides equaUy or only slightly better than with nucleotides (Fig. 1). They also react well with polymers in which the bases are accessible, such as denatured DNA. When the periodate oxidation technique (with ring breakage and later reduction) is used for immunogen preparation, the resulting antibodies react slightly better with deoxyribonucleosides than with ribonucleosides. 

A valuable adjunct to the periodate oxidation technique is the Barry degradation, which can give information on the sequence of units in a polysaccharide. It is particularly useful in assigning a single structure to a polysaccharide from several possible structures which may be proposed from... 

Other methods of identification include the customary preparation of derivatives, comparisons with authentic substances whenever possible, and periodate oxidation. Lately, the application of nuclear magnetic resonance spectroscopy has provided an elegant approach to the elucidation of structures and stereochemistry of various deoxy sugars (18). Microcell techniques can provide a spectrum on 5-6 mg. of sample. The practicing chemist is frequently confronted with the problem of having on hand a few milligrams of a product whose structure is unknown. It is especially in such instances that a full appreciation of the functions of mass spectrometry can be developed. 

The application of periodate oxidation to a problem can best be considered in terms of an approach from two separate standpoints. The first generally involves the application of analytical techniques to semimicro quantities of material, and sometimes suffices to yield the desired information. After performing this preliminary, analytical investigation, the second or preparative phase may be undertaken. This entails use of much larger amounts of material, with the intention of isolating the end products of oxidation. If these are obtained, their properties should serve to confirm the results of the preliminary, analytical work and, if this is proved correct, the method is then available as a route to the desired synthetic compound. 

A different technique utilizes the Warburg respirometer to measure the carbon dioxide liberated by formic acid from a dilute, sodium bicarbonate solution.84 286 Successful application of the method necessitates a pH value of 5.7, which is somewhat high for optimal periodate oxidations, but good results were reported in experiments on a number of materials, including polysaccharides. The procedure is applicable to very small amounts and does not require prior destruction of periodate. 

Table I shows the results of periodate oxidation and methylation applied to amylopectins from various sources. If the percentage of amylose in a whole starch is known, the length of unit chain of the amylopectin component can be calculated from the results of periodate oxidation of the whole starch (see Table II). The length of unit chain appears to depend on the botanical species, but not the variety, from which the starch wras isolated. In the case of tapioca and corn amylopectins, sub-fractionation of these by precipitation with methanol, followed by periodate oxidation, showed the sub-fractions had the same degree of branching as the original amylopectins.71 The action of periodate on whole starches and amylopectins is now so well established and accurate, that it may well completely supersede the methylation technique for pure starches, in view of its many advantages.

Structural investigations into the degree of branching and into the position and nature of glycosidic bonds and of non-carbohydrate residues in polysaccharides may include periodate oxidation and other procedures such as exhaustive methylation. X-ray diffraction and spectroscopic techniques such as nuclear magnetic resonance and optical rotatory dispersion also give valuable information especially relating to the three-dimensional structures of these polymers. 

Unsaturated acids may be split chemically at their double bonds. Permanganate-periodate oxidation has been used to produce the corresponding carboxylic acids, while an alternative technique of ozonolysis results in the formation of aldehydes and aldehyde esters. All these reaction products may be identified by GLC and the information used to determine the position of the double bond in the original fatty acid. 

This C-13 n.m.r. analytical technique has been referenced against 27 dextrans which had been studied by periodate-oxidation and permethylation fragmentation g.l.c.-m.s. techniques (14). 

In examining the structure of a polysaccharide, it is convenient to consider the methods involved under the three main headings (a) quantitative analysis, (b) methylation, and (c) periodate oxidation. These techniques may be supplemented by partial or enzymic hydrolysis as the circumstances indicate. Each of these aspects of polysaccharide chemistry may be aided by the application of gas-liquid chromatography, either qualitative or quantitative, or both. Thus, separations impossible by other techniques may often be achieved, and analytical data obtained in a fraction of the time demanded by other methods. 

Potentiometric techniques have been used to study autonomous reaction rate oscillations over catalysts and carbon monoxide oxidation on platinum has received a considerable amount of attention43,48,58 Possible explanations for reaction rate oscillations over platinum for carbon monoxide oxidation include, (i) strong dependence of activation energy or heat of adsorption on coverage, (ii) surface temperature oscillations, (iii) shift between multiple steady states due to adsorption or desorption of inert species, (iv) periodic oxidation or reduction of the surface. The work of Sales, Turner and Maple has indicated that the most... 

As regards the l,l-bis(acylamido)-l-deoxyalditols, the first structural study was that of Fletcher and coworkers,69 who conducted oxidations with lead tetraacetate. These authors postulated an empirical relationship between the position of the oxidation curve in a graph and the number of hydroxyl groups present. They compared the curves afforded by a series of l,l-bis(acetamido)-l-deoxyalditols with those of alditols having the same number of free hydroxyl groups, and observed a close correspondence. At present, periodate oxidation is widely applied to these mono- or di-saccharide derivatives. Under controlled conditions, the methylation technique can also be applied.42... 

The periodate oxidation of carbohydrates, an important analytical technique, has been reviewed earlier in this Series.73 The dialdehydes obtained on periodate oxidation of polysaccharides have also been discussed.74 The requisite for the degradations to be discussed here is that part of the sugar residues in a polysaccharide are not oxidized by periodate and can be obtained separated from the oxidized residues as mono-, oligo-, or poly-saccharide derivatives after some chemical treatment. Characterization of these products may then give significant structural information. 

The uniqueness of the aldofuranosides, as compared to the aldopyrano-sides, was recognized initially through the difference in rates of hydrolysis by acid. This difference was confirmed by the use of methylation techniques the latter have, to a great extent, been supplanted by the periodate oxidation of Jackson and Hudson120 and by oxidation with lead tetraacetate. Hudson s rules of isorotation were also employed to a great extent. 

The structure of umbelliferose (3) was elucidated 4 by a combination of enzymic (using a-D-galactosidase) and chemical techniques (using periodate oxidation, methylation, and acid hydrolysis). 

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