Paul F. Pilch a n d Ronald L. Somerville Depanment of Biochemistry Purdue University West Lafayette, Indiana 47907
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Periodate Oxidation of Vicinal Hydroxyls Applications to student experiments
Periodate is a wwerful and s~ecificoxidizine reaeent widelv employed in preparative and analytical carbohydiate chemistry ( 1 I. In teachinp. laboratories. oeriodate rlenvare followed by &metric determination of foimic acid has been used t o study the degree of branching of complex carbohydrates such as glycogen (2, 3). Periodate oxidation procedures recommended for use in student l a b have not included quantitation of the amount of periodate consumed, in large part because either titrimetric (4) or spectrophotometric methods (5) commonly employed are relatively slow, insensitive, and cumbersome. We describe a quick, sensitive method which is readily adaptable for quantitating periodate consumption in a variety of undergraduate laboratory teaching contexts. Avigad (6) described the rapid stoichiometric bleaching by periodate of the violet ferrous 2,4,6-tri-2-pyridyl-s-triazine (TPTZ) comolex (Am.. . ... . 593 nm) to a colorless comoound. kvigad (6) f o k d the molar extinction coefficient for bleaching to be 37,000. This value was confirmed in our studies. Bleaching can be accurately quantitated with inexpensive s~ectroohotometerssuch as the Bausch and Lomb "Snect k i c i0." Only moderate amounts of sugars and periodate in the ranre of 5-200 nmoles are needed. several';ypes of experiments based on this procedure can he msils desirncd. Studcnts can be asked to verifv the stoichiomeiry of'the oxidation reaction (one mole-periodate consumed per mole of vicinal diol)
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Second-order plot of the reaction of perlodate with propylena glycol ( 0 ,k = 135 M-' s-')and with (D.Lttdeoxy-t-fluaroglycerol(0, k = 5.75 M-' s"). The reactions were conducted al-5%. 2 m M in ID4, 1 mM in diol, and 50 mM in acetate buffer, pH 5.0. At the intervals noted 0.1ml of reaction minure was added to 4.9 ml undiluted TPTZ reagent. The resultingsbswbancyvalues at 593 nrn were used to compute the conceohatians of diol and periadete at each
time. periodate to strong light should he avoided. Fresh solutions should he prepared weekly. Reaction mixtures of periodate and sugar can be prepared in a variety of concentrations and final volumes depending on the specific experiment. For determining the stoichiometry of oxidation, 2.0 ml of solution containing acetate buffer, pH 5.0 (50 mM), NaIOd (20 mM), and suear (2-10 mM) were oreoared. . . At timed intervals after initial mixmg. 0.1 ml of aquirkly prepared 1:2Ud1lut1ontin H z 0 1 d t l w reaction mixture uas added to 4.9 ml 'I'PT7. macent, prr, iously adjusted togivean Asslof- 0.9 witha water blank. T h ~ procedure r is repeated at various intervals until a constant A593 value is reached. After periodateand TPTZ have been mixed, the unhleachedcolor is stahle and can he read at any convenient time. Knowing the extinction coefficient,the student then calculates the amount of periodate remaining and the amount consumed by difference. When the kinetics of reaction are to be monitored, the reaction temperature should be held constant. Values from ambient to 3I0C proved satisfactory for the previously mentioned glycosides. Far acyclic dials, zero degrees (ice bucket) as well asa 10-folddilutionof reactants was found to be necessary. We found 2 mM 104- and 1 mM glycol (the fig.) to he convenient although excess glycol could have served equally well. The amount of reaction mixture added to the TPTZ reagent should hecontrolled so that complete bleaching does not occur. The above procedure is readily adaptable for measurements of reaction rates as a function of temperature. Activation energies may then he determined from an Arrhenius plot. Reaction rate can also he determined as a function of hydrogen ion concentration; pH is known to he an important parameter in periodate oxidations (I). ~
Commercially available simple polyols such as sorbitol or inositol react completely a t r w m temperature in less than one hour with the conwmption of five o; six moles of periodate per mole of sugar, respectively. Conformational effects on the mte of periodate-sugar reaction can readily be observed. For example, the enantiameric glycosides, methyl-n-D-glucopyranoside and meth\rl-n-D-mannou\~anosidediffer markedlv in initial rate of oxidation (6). The oxidation of acyclic glycols is fast compared to reducing sugars. Cleavage of cis-hydroxyls is faster than that of trans-hydroxyl groups. Finally, one can nerform kinetic exneriments with anorooriate diols and deiermine second-order rate constants ? b e kg.). The illustrated e x ~ e r i m e nemnlovs t a non-commercial diol.. (D.L)-l-deoxv. .. 1-fluoroglyceroi: ~ i w e v e ar variety of readily available such as 1-phenyl-12-ethanediol, 3-chloro-1, 2-propanediol, and 2,3-butanediol should provide interesting, instructional results. .A
Experimental TPTZ reagent is prepared by dissolving 75.0 mg TPTZ (Aldrich) in 46.0 ml glacial aceticacid. After adding 210 mlof 1 N sodium acein 100mlH20to theTPTZ tate and 31.4 mgof Fe(NH4)2(S04)&H20 solution, the mixture is made up to 1 I with water (6).This solution has an A6g3 of -1.8 in 1-em cells and may be diluted with 1M sodium acetate to a convenient absorbance. It is stable for s t least a month. Sodium metaperiodate stock solution (50 mM) is prepared by dissolving 1.0695 g in 100 ml H20 (volumetric flask). Expasure of
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Literature Cited (1) Stanek, J.. Cmw. M. Komumk. Land P a d , J.,'Tho Monosemharidn:'Aeademic Press. New York, 1363, p. 903. (2) Redins, G.,'"Experimental Methala in Birrhernirtry,"3rd Ed., W. 0. Saunders Co, Philadebhis. 1971. p. 161. (3) P1ummer.D. T.,"Anlntroduetion toPracticalBi~hrmistry:'Mffiraw-HillBookCo., London, 1971,p. 121. (4) Dyer. J.R..MefhodsBiaehem.Anol., 3,111 11956). ( 5 ) Dixon, J.S.,sndLipkin,D..Anal.Chem., 2LlM2 (19%). (6) Avigsd,
G..Corbohyd. RGB 11.119 11969).
Volume 54, Number 7, July 1977 1 449
