Periodate oxidation of HRP

Figure 20.14 Periodate oxidation of HRP creates aldehyde groups on the carbohydrate chains of the enzyme. Reaction with a Fab fragment then may be done using reductive amination to produce a lower-molecular-weight complex than would be obtained using intact IgG antibodies.

The protocols for periodate oxidation of HRP and its conjugation with other proteins may be found in Chapter 20, Section 1.3. 

The use of periodate coupling chemistry for HRP first was introduced by Nakane and Kawaoi (1974 see also Nakane, 1975). In the first step of their protocol, the few amine groups on HRP were initially blocked with 2,4-dinitrofluorobenzene (DNFB) before periodate oxidation. 

Conjugation of Periodate-Oxidized HRP to Antibodies by Reductive Animation... 

The following protocol assumes that HRP has already been periodate-oxidized by the method of Section 1.3. 

Acyl nitroso compounds (3, Scheme 7.2) contain a nitroso group (-N=0) directly attached to a carbonyl carbon. Oxidation of an N-acyl hydroxylamine derivative provides the most direct method for the preparation of acyl C-nitroso compounds [10]. Treatment of hydroxamic acids, N-hydroxy carbamates or N-hydroxyureas with sodium periodate or tetra-alkyl ammonium periodate salts results in the formation of the corresponding acyl nitroso species (Scheme 7.2) [11-14]. Other oxidants including the Dess-Martin periodinane and both ruthenium (II) and iridium (I) based species efficiently convert N-acyl hydroxylamines to the corresponding acyl nitroso compounds [15-18]. The Swern oxidation also provides a useful alternative procedure for the oxidative preparation of acyl nitroso species [19]. Horseradish peroxidase (HRP) catalyzed oxidation of N-hydroxyurea with hydrogen peroxide forms an acyl nitroso species, which can be trapped with 1, 3-cyclohexanone, giving evidence of the formation of these species with enzymatic oxidants [20]. 

Immediately purify the oxidized enzyme by gel filtration using a column of Sephadex G-25. The chromatography buffer is 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.2. To obtain efficient separation between the oxidized enzyme and excess periodate, the sample size applied to the column should be at a ratio of no more than 5 % sample volume to the total column volume. Collect 0.5-ml fractions and monitor for protein at 280 nm. HRP also may be detected by its absorbance at 403 nm. In oxidizing large quantities of HRP, the fraction collection process may be done visually—just pooling the colored HRP peak as it comes off the column. 

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