Activation of Enzymes with Sodium Periodate

Enzymes that are glycosylated (i.e., HRP and GO) may be oxidized according to the following method to produce aldehyde groups for reductive amination coupling to an antibody molecule. 

Dissolve sodium periodate in water at a concentration of 0.088 M. Protect from light. 

Immediately add 100 pi of the sodium periodate solution to each ml of the enzyme solution. This ratio of addition results in an 8mM periodate concentration in the reaction mixture. Mix to dissolve. Protect from light. 

React in the dark for 15-20 minutes at room temperature. If HRP is the enzyme being oxidized, a color change will be apparent as the reaction proceeds—changing the brown-ish/gold color of concentrated HRP to green. Limiting the time of oxidation will help to preserve enzyme activity. 

Pool the fractions containing protein. Adjust the enzyme concentration to lOmg/ml for the conjugation step (see next section). The periodate-activated enzyme may be stored frozen or freeze-dried for extended periods without loss of activity. Do not store the preparation in solution at room temperature or 4°C, since precipitation will occur over time due to self-polymerization. 

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 poohng the colored HRP peak as it comes off the column. 

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The reaction mixture contained in a final volume of 0.1 mL, 0.1 M sodium phosphate buffer (pH 6.8), 1.0 mM tryptamine, 5 mM secologanin, and 3 mM dithiothreitol. To inhibit glucosidase activity, 100 mM D(+)-gluconic acid-5-D-gluconolactone was included. The incubation was started by addition of 10 /xL of enzyme. After 30 minutes of incubation at 30°C, the reaction was stopped by addition of 0.1 mL of 5% trichloroacetic acid. Before centrifugation, 25 /xL of 8 mM codeine hydrochloride was added as the internal standard. HPLC analysis was performed on 4 /xL aliquots. With enzyme purified to a specific activity of 710 pkat per milligram of protein, the reaction was linear with time for 1 hour and with protein up to at least 50 /xg of protein during a 20-minute incubation period. 

Replacement of side chain carbon C21 by thioester sulfur retains corticoid activity and at the same time provides a function that is destroyed by serum enzymes. The synthesis starts with the potent antiallergic agent flumethasone (32-1). Reaction of that steroid with periodic acid cleaves the terminal bond in the hydroxyacetone side chain to give the hydroxyl acid 32-2 (Scheme 7.32). Steric hindrance around the acid invoke the need for extra activation of that function. Reaction of 32-2 with diphenyl chlorophosphate thus provides the mixed anhydride 32-3. This intermediate is not isolated but reacted in situ with AA -thioformamido chloride. The transient new mixed anhydride, 32.4, then undergoes an internal O to S rearrangement to give the acyl thioacid 32-5. Saponification with sodium hydroxide affords the corresponding thioacid. Alkylation of that intermediate with fluoromethyl bromide then yields the fluoromethyl thioester fluticasone (32-6). 

The effects of oscillating electric fields on ion accumulation processes are also explained by SCM calculations (20). The oscillations lead to periodic changes in the ionic concentrations that are functions of the frequency, but the percentage change is greatest in those concentrations with the lowest steady-state values. In particular, sodium on the inner surface and potassium on the outer surface show maximal changes at about 100-200 Hz. These two ionic concentrations normally control the activity of the Na, K-ATPase of cell membranes, and increases could stimulate the enzyme. 

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