Hi all.
I ran the enzyme activity (pure Tyrosinase after purification using recombinant e coli) using L-Dopa with this condition
50 mM phosphate buffer ph 6,
1mM L-Dopa
25 C temperature
0.5 mM sls for enzyme activation
Result was positive with appearance of pink solution.
I did kinetic parameter with specific activity, which all show positive result.
However, when i started to do different temperature activity, now there was no colour change ? So, i repeated the same activity test also no colour change.
So, has anyone experience the same ? Attached is the activation test that showed pink solution.
Tyrosinase is inactivated during the DOPA-tyrosinase reaction and the reduction in activity is proportional to the time.
Tyrosinase inactivation can also occur when ascorbic acid is added to the reaction system; in which dopaquinone is converted back to DOPA by preventing melanin formation.
Hi Niti Sharma , thank you for your response. The issue here is that the tyrosinase was active before with the conditions mentioned. However, with the same conditions, there was no activity when we repeated the assay. same protein concentrations and same condition of assay.
Hi Dr. Adam B Shapiro . Both were stored in -20 C. actually i just noticed when i rerun the same sample after the assay on sds page(no noticeable band ). so, I can say that the protein after dialysis was degraded, maybe. However, we used the same dialysis buffer which was 50 mm Tris HCL pH 8 (NEW STOCK from the successful assay previously). It was just confusing because all conditions were the same.
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