Table 1.
Activation of horseradish peroxidase suspended in 97% acetone by various excipients colyophilized with the enzyme
| Entry | Excipient* | Activation effect† |
|---|---|---|
| 1 | o-Methoxyphenol (guaiacol) | 4.0 |
| 2 | o-Methylphenol (o-cresol) | 8.0 |
| 3 | p-Methylphenol (p-cresol) | 7.7 |
| 4 | o-Aminophenol | 15 |
| 5 | o-Hydroxybenzoic acid | 18 |
| 6 | o-Hydroxybenzyl alcohol | 62 |
| 7 | m-Hydroxybenzyl alcohol | 15 |
| 8 | p-Hydroxyphenethyl alcohol | 10 |
| 9 | Aniline | 5.8 |
| 10 | p-Aminobenzoic acid | 20 |
| 11 | p-Methoxyaniline (p-anisidine) | 6.6 |
| 12 | Benzyl alcohol | 4.0 |
| 13 | Benzoic acid | 16 |
| 14 | p-Nitrophenol | 15 |
| 15 | Benzylamine | 7.0 |
| 16 | 1-Phenyl-1,2-ethanediol | 18 |
| 17 | trans-1,2-Cyclohexanediol | 17 |
| 18 | cis-1,2-Cyclohexanedicarboxylic acid | 4.7 |
| 19 | Cyclohexylamine | 4.0 |
| 20 | PEG | 6.5 |
| 21 | Sucrose | 4.0 |
| 22 | Tris | 27 |
The enzymatic activity in all instances was measured in the oxidation of 25 mM guaiacol with 0.25 mM hydrogen peroxide. The reaction medium, in which these substrates were dissolved, was formed by mixing an aqueous acetate buffer (10 mM, pH 5.0) with anhydrous acetone in a 3:97 (vol/vol) ratio. HRP was lyophilized from an aqueous buffered solution containing a given excipient as described in Materials and Methods, suspended in the acetone medium at 10 mg/ml, briefly sonicated, and stirred at 25°C. For other experimental conditions, see Materials and Methods.
The concentration of all excipients in the aqueous solution of HRP before lyophilization was 100 mM (on the monomer basis in the case of PEG) except for o-aminophenol which, being insoluble at this concentration, was 40 mM). For two of the excipients listed, o-hydroxybenzyl alcohol and Tris, we examined the dependence of the activation effect on the excipient concentration in the range from 20 to 400 mM. It was found that the maximal effect was attained at a 100 mM excipient concentration in the aqueous HRP solution before lyophilization, presumably because of a physical blockage of the active sites in the enzyme suspension by the excipient molecules at higher concentrations (2).
Defined as the initial rate of the enzymatic peroxidation catalyzed by HRP lyophilized in the presence of an excipient divided by that catalyzed by HRP lyophilized in the absence of excipients. Several measurements conducted in duplicate revealed that the experimental error was typically in the 5–15% range and never exceeded 25%.