DMAB/MBTH

Fig. 14.1. Determination of the optimum dilution of H202as substrate with horseradish POase C with DMAB/MBTH as H-donor. High dilutions as well as high concentrations of the substrate decrease the detectability of the enzyme. The optimum is around 0.0025-0.003% H2O2 (reproduced from Tijssen et al., 1982 courtesy of Archives...

Fig. 14.3. DMAB/MBTH as H-donor and the eflect of H2O2 as compared to 5-aminosalicylic acid (D). In the left frame various concentrations of H2O2 namely 0.03% (A), 0.0003% (B) and 0.003% (C) were used with the standard concentrations of DMAB (25 mg/100 ml) and MBTH (0.75 mg/l(X) ml). The response could be increased (right frame) by increasing the DMAB/MBTH concentration two-fold (F) and four-fold (G), whereas a two-fold dilution decreased the detectability (E),the H2O2 concentration being kept constant (0.003%). The DMAB/MBTH system gave, however higher backgrounds. Courtesy Archives of Virology.

Determination of POase activity in solid-phase EIA is different from that of free POase in solution (Section 10.1.1.5). The amount of chromophore produced during a certain period (20 min, 30 min, 1 h, etc.) is determined from the absorbance [5-AS at 492 nm ABTS at 415 nm OPD at 492 nm if acidified with HCI (pH 1.0), at pH 5.0 the peak is at 445 nm (usual filter 436 nm) ODA at 400 nm DMAB/MBTH at 590 nm (usual filter, 620 nm)]. To obtain the exact color development in each sample, it is necessary to stop the reaction after identical periods. Color development with ABTS and 5-AS can be stopped with 0.2 volume of 37 mM NaCN (poison ), oxidation of OPD is blocked with 0.5 volume of 4 M H2SO4 or HCI and the reaction with MBTH/DMAB is arrested with 0.1 volume of IM H2SO4 or HCI (the blue-purple color changes to blue, but the absorption at 590 nm remains virtually unchanged). 

For oxalate detection, authors proposed a similar detection approach for recognition of oxalate via an immobilized oxalate oxidase/peroxidase couple and dye precursors MBTH (3-methyl-2-benzothiazolinone hydrazone) and DMAB (3-dimethylaminobenzoic acid). The peroxide generated by oxidation of oxalate to CO2 reacted with the dye precursors in a peroxidase-catalyzed reaction to yield an indamine dye with absorption maximum at 590 nm. The concentration of oxalate was correlated with increased absoiption from dye. 

A sensitive and versatile chromogenic assay for POase is based on the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethylamino)benzoic acid (DMAB) (Ngo and Lenhoff, 1980) in the presence of substrate. MBTH is the donor which, after oxidation, reacts with DMAB to form a cationic in-... 

ODA), 2,2 -azino-di-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), o-toluidine (OT), o-phenylenediamine (OPD), 3,3, 5,5 -tetramethylben-zidine (TMB), and the MBTH-DMAB pair [MBTH 3-methyl-2-benzothiazolinone hydrazone DMAB 3-(dimethyl-amino) benzoic acid]. The preparation of the most suitable H-donors is given in Table 14.13. 

Just before use, mix 7 volumes of 100 mM sodium phosphate-citrate buffer, pH 6.0, with 1 volume of the MBTH and 1 volume of the DMAB solutions. 

Oxidative coupling of MBTH and DMAB (Section 10.2.1.4.2) has been applied successfully for ElA (Tijssen et al., 1982 Geoghegan et al., 1983). The detection limit for POase is about 2 fmoles and both detectability and background levels are higher than with 5-AS... 

Tijssen et al., 1982) (Fig. 14,3). Geoghegan et al. (1983) found large differences among the buffers used with these H-donors 100 mM phosphate-citric acid, pH 6.0 was as satisfactory as HEPES, but much cheaper. MBTH and DMAB are poorly soluble below pH 5.5-5.8, but higher pH or lower ionic strength increases the absorbance of the blanks. The reaction should be stopped (by enzyme inhibition) after 20-30 min. Both DMAB and MBTH are photosensitive and the blanks develop color when exposed to light. 

Glucose + oxygen—> gluconic acid + HjOj HP2 + MBTH -I- DMA >BMBTH-DMAB (blue)... 

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