@ Soudabeh, please have a look of the attached file.

The iron content is directly related to the hemin content assuming no other sources of Fe. ICP or AAS could be used to determine the Fe concentration. Alternatively colormetric routes could be used.

hi

I think you should know the initial concentration of hemin, Then read the OD of residual buffer from the centrifuge step and calculate the extinction coefficient formula to assay the concentration of hemin unbound in buffer .

"The concentration of hemin was determined at 385 nm using an extinction coefficient of 5.84 × 104 cm−1 m−1"

https://www.ruf.rice.edu/~bioslabs/methods/protein/abs280.html

https://pdfs.semanticscholar.org/1b99/193db15785006b0afde9ce5483abfe304d5b.pdf

https://www.princeton.edu/~hecht/papers/1997%20rojas%20de%20novo.pdf

https://www.jenabioscience.com/images/76f5fe1aed/ML-108_MSDS.pdf

good luck

K672-100 Heme Colorimetric Assay Kit

https://www.biovision.com/documentation/datasheets/K672.pdf