While studying kinetics for HRP with TMB as a substrate, my reaction mixture rapidly turns yellow after turning blue for a very short while. Also, the absorbance taken with 30s interval went up for approx. 1 min and then started to fall down. Because of this, I am unable to draw linear Abs/time plot to find initial velocity of reaction. What could be the reason for this, can anybody guide me please?
This reaction leads to a complex mixture of species. Perhaps this paper may be a good start for your literature study.
http://www.jbc.org/content/257/7/3669.full.pdf
The fast change to yellow may indicate that your HRP concentration is much too high. Please consider that this reaction is extremely sensitive. HRP might be detected down to 10-15 mol/L.
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