Hello, I have some problem in my protein and humic acid determination method using Lowry method. I would really appreciate any suggestion for the Lowry method and thank you in advanced for your answer.

In my experiment, I've extracted EPS fraction of microalgae culture and would like to quantify protein, humic acid, polysaccharide.... About the protein and humic acid quantitative method, I've found some articles said that modified Lowry method can be used (reference [2], attached link).

For my understanding, total absorbance at 750nm (with the addition of CuSO4) will be the total absorbance of protein, humic compound, chromogenic amino acid... and the blind absorbance at 750nm (without the addition of CuSO4) will be the absorbance of tyrosine, tryptophan content [1], humic compound, chromogenic amino acid....And when the sample have high concentration of these compounds, conventional Lowry method tends to over-estimate the protein concentration.

In the attached articles, the authors said that without CuSO4, absorbance of bovine serum albumin (BSA) decreased to 20% and hence, that number was used to calculate the Aprotein and Ahumic in formula 3 and 4. But when I make the standard curve (with and without CuSO4 addition) (attached picture), I found the decrease to 20% is not always true but it depends on the range of BSA concentration.

So, I wondering should I just apply the formula 3 and 4 for calculation of protein and humic acid, or I should re-calculate the absorbance decrease at my sample protein concentration ? 

I'm afraid my question is a little complicated to understand, so I've tried my best to draw it in detailed in the attached picture.

Or if there is any alternative simple method for protein and humic acid determination, would you please suggest me. Thanks in advanced for all of your answers.

Reference:

[1] Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J biol Chem, 193(1), 265-275

[2] Fr, B., Griebe, T., Nielsen, P. 1995. Enzymatic activity in the activated-sludge floc matrix. Applied Microbiology and Biotechnology, 43(4), 755-761. (attached link)

http://link.springer.com/article/10.1007/BF00164784