I have been trying to develop an assay to measure recombinant hylauronidase activity. It is a chromogenic method which measures N-acetylglucosamone (NAG) production by hyaluronidase on hyaluronic acid.
We have hylaronidase in sodium formate buffer, add hylaronic acid for 150 minutes at 37 C, then we add 2M potassium tetraborate, boil for three minutes, cool and add pDMAB. We get a lovely purple colour which we measured at 585nM. We calibrate off a known amount of NAG standard curve. We are trying to measure rec hyaluronidase spiked into plasma which has its own endogenous hyaluronidase, which has to be measured and subtracted.
Problem is the colour fades really quickly after addition of pDMAB, so it is really difficult to standardise the technique.
Has anyone used this method and have any tips? I am not a chemist and have picked up the method from reading papers.
Hi Vivek, thanks for your input. Unfortunately I cannot access that paper without a subscription. The 2M potassium tetraborate was supposed to help with the turbidity produced by serum proteins (ref reinvestigated Reissig method; Nagaraju et al, Clin Lab Sci 2011; 24(3);172).
I went back and read the original Morgan Elson paper of 1934 and found some help in there - the concentration of HCl in the pDMAB reagent is important in the colour development. pDMAB is dissolved in acetic acid/Hcl, at 10% HCl, as we were using, the colour is maximal straight away and then fades rapidly, adding only 5% HCl, the colour develops over 20 minutes to maximal, then slowly fades, with less HCl, colour formation is really slow.
We tried a little experiment yesterday afternoon, and it worked well, so we have changed the concentration of HCl in our pDMAB reagent.