Why guaiacol turn to brown eventhou there is no enzyme added during assay when i am using buffer Tris-HCl and sodium acetate?
in my understanding, when there is brown color appearance, it means that guaiacol have been oxidized by enzyme laccase.
but in my case, before i proceed my enzyme assay with addition of enzyme, i have tested it first all the component without additional of enzyme
so here are my reaction component
SET 1 (CONTROL ENYZME)
1. SUBSTRATE (GUAIACOL)
2. BUFFER (SODIUM ACETATE)
3. METAL IONS (COPPER SULPHATE)
RESULT: TURN TO BROWN COLOR
SET 2 (CONTROL ENYZME)
1. SUBSTRATE (GUAIACOL)
2. BUFFER (TRIS-HCL)
3. METAL IONS (COPPER SULPHATE)
RESULT: TURN TO BROWN COLOR
SET 2 (CONTROL ENYZME)
1. SUBSTRATE (GUAIACOL)
2. BUFFER (SODIUM PHOSPHATE)
3. METAL IONS (COPPER SULPHATE)
RESULT: NO CHANGE
I am confuse because many paper propose to used sodium acetate for laccase assay. But it turn out it give different result when i do it and it create FALSE POSITIVE result if i keep use SODIUM ACETATE AS BUFFER
KINDLY HELP ME TO CLARIFY THIS PROBLEM
THANKS IN ADVANCE :)
Add guaiacol before addition of enzyme as guaiacol is phenolic molecules and when exposed to light and air it becomes dark in color.
Dear Siti NABILAH Mohamad Shudirman,
The guaiacol has a high affinity for oxygen, and it binds instantly with oxygen to form tetraguaiacol, which is brownish in color.
Dear Siti. The main cause for this is a mold growing in the buffer. Can you produce your own buffer starting from pure drgs? another possibility In our lab we experienced laccase-mimicring activities by glass cleaning products (even non enzymatic ones). I would suggest to rinse the glas tubes with distilled water to get rid of any detergent remnants. Also light might play a role, as Trupti mentioned above. Good Luck!
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