How large was the absolute value of your "negative" absorbance? Could you give example values?

Morning, sorry for the late reply.

Example:

Blank (substrate + buffer) : -0.0001

sample (buffer + sample + substrate) :

min absorbance

1 -0.278

2 -0.269

3 -0.256

4 -0.248

I had tried for a week with some modification, then the results are still negative. However, last Friday I did tried to change the blank, I mixed the buffer with the sample, surprisingly, the blank reading was 0.000 and the absorbance of my sample was also positive. Nevertheless, even though I able to re-zero, I'm wondering why it was happen that way. For all the journals that I read the blank should be the substrate plus buffer. So is anyone know why when the re-zero with sample, the value is 0.000 but -ve if using the correct blank...

Likely, when you are adding the ycw,you are diluting the buffer and substrate concentrations so that they are lower than your blank. This will cause a negative absorbance. You need to adjust the final concentrations of the buffer and the substrate to be the same for the blank and the sample (i.e., higher concentrations of buffer and substrate should be added to the sample so that when you add the extra volume of ycw, their concentrations are the same as the blank).

thanks mr brian for the suggestion. I got the answer already, I have to ensure the correct type of cuvette. Before this I used the quartz cuvette for the wavelength of 425nm, but now I realized that I should use the plastic cuvette for the wavelength above 335nm. Actually the original method is by measurement changes of absorbance values using 425 nm. So now I can proceed with the original method successfully. Thank you.