I'm running OUC enzyme assays on dogfish intestinal tissues. I'm having trouble with the OTC enzyme assay in particular. I know my protocol works as the positive control (cow liver) shows no colour at time zero (i.e. killing the reaction with 70% PCA before it begins) and a dark yellow after allowing the reaction to run for 10 min and then killing it with 70% PCA, and finally assaying both time points for citrulline.

For the dogfish intestine I cannot get a colour change (i.e. difference in citrulline produced). Time zero has the same absorbance (at 412 nm) as after allowing the OCT reaction to run 10 min (or 20, or 30 min). The colour does darken with increased volumes of sample (supernant) but never a difference between time zero or stopping it after 10, 20 or 30 min. In fact the time zero tends to have a slightly higher absorbance (darker colour) than after 10, 20 or 30 min.

I have tried various volumes of tissue (supernant; 10, 25, 50, 75, 125ul) for the OCT assay, I've allowed the reaction to run longer (10, 20 and 30 min), I've doubled the concentration of the OCT cocktail, I've increased the volume of PCA to 4x the required volume, I've used homogenization buffer with and without glycerol, but nothing seems to bring about a separation in absorbance values between time zero and later time points.

Any suggestions?