The azoalbumin proteolysis reaction should produce an absorbance increase. Therefore, the blank absorbance should be lower than the reaction absorbance. It would be helpful to know more about the composition of the blank and sample.

Thank you for the answer. I prepared 0.6% azoalbumin in Tris-HCl pH 8.5 with addition 50% urea. The reaction mixture composed from 1 ml of 0.6% azoalbumin and 1 ml of cell suspension after homogenization. The blank was without cell suspension. After 3.5 h incubation at 37°C, reaction was stopped by addition 0.3 M TCA. Nevertheless, I found several synthetic substrates in our labs and finally I decide to use some of them.

Why do you use 50% urea in this reaction mixture? Also after you add TCA (to remove the unreacted substrate by centrifugation),I guess you are referring to the absorbance of the solubles (i.e supernatant fraction containing the digested azopeptides) isn't it? Since it is an azosubstrate, are you not adding 10N NaOH to develop the colour (read at 440 nm)? The NaOH neutralizes the TCA. Even at the end of these steps, does your intracellular enzyme fraction show a negligible absorbance vis-a-vis blank? In which case, maybe your intracellular enzymes show a different substrate specificity unlike the extracellular fractions which cleave albumin.