P- Acetamidobenzyl

The spectrophotometric determination of the reagent equivalents bound to the enzyme is based on the assumption that the spectrum of the alkylated enzyme results from the addition of adenosine deaminase and 9-(p-acetamidobenzyl) adenine spectra. The spectra have two isosbestic points at 244 nm ( = 21.1 X 10 Af- cm" ) and at 265 ( = 21 X 10 cm" ). The adenine derivative exhibits maximum absorbance at... 

When the enzyme is mixed with 9-(p-acetamidobenzyl) adenine a difference spectrum is observed nevertheless the differences are small (lower than 1% at the considered wavelengths) in comparison to the absorbance of the reagent and the enzyme and are therefore disregarded. 

Figure 2. Preparing BLEDTA from bleomycin A2. The bifuntional chelating agents (S)-l-(p-bromo-acetamidobenzyl)-EDTA and racemic l-(p-bromoactamidophenyl)-EDT A have been used in this procedure (25).

To remove the unreacted 9-(p-bromoacetamidobenzyl) adenine the reaction mixture is passed through a column (0.9 X 10 cm or 2.5 X 10 cm) of Sephadex G-25 medium equilibrated with 0.1 Af sodium phosphate at pH 8, or 0.1 M NH4HCO3. The major part of the reagent precipitates on the column. Alternatively, the reaction mixture is maintained at 0° for 60-120 min before column separation during this period no further inactivation of the enzyme is observed and a great part of the reagent precipitates. The latter procedure is preferred when tritiated 9- (p-bromo-acetamidobenzyl) adenine is used. In this case Sephadex G-25 columns (0.9 X 20 cm or 2.5 X 20 cm) equilibrated with 10% dimethyl sulfoxide are far more useful. 

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