ABSTRACTThe interaction between bovine hemoglobin (BHb) and palmatine hydrochloride (PMT) was investigated at different temperatures using multispectroscopy, as well as the effect of common metal ions (Ca2+, Mg2+, Zn2+, Cu2+, Fe2+, Fe3+, Co2+, Ni2+) on the BHb–PMT system. Results showed that the quenching mechanism of PMT on BHb was a static process. The electrostatic force played an important role in the conjugation reaction between BHb and PMT. The order of magnitude of the binding constants (Ka) was 104, and the number of binding sites (n) in the binary system was ~ 1. The binding distance (r) was ~ 2.44 nm and the primary binding for PMT was located at β‐37 tryptophan in the hydrophobic cavity of BHb. In addition, the Hill's coefficients were ~ 1. Synchronous and circular dichroism spectra revealed that the microenvironment and the conformation of BHb were changed during the binding reaction. Copyright © 2013 John Wiley & Sons, Ltd.