Abstract: OBJECTIVE To optimize the conditions of polyethylene glycol (PEG)-modified rhk2a reaction and research the pharmacodynamic characteristic of the modified mPEG-rhk2a. METHODS By using mPEG-ALD with weight of 5 000, the rhk2a was modified on its N-terminal amino. Used single factor test and orthogonal experiment to optimize the conditions of modification and established an HPLC detection method to assay the protein concentration of mPEG-rhk2a. The positive inotropic effect of mPEG-rhk2a in guinea pigs was investigated. RESULTS Optimized conditions of reaction were as follows: when the pH was 5.0, the rhk2a/mPEG(molar ratio)=1∶20, the reducing agent was 30 μL, the reaction time was 12 h, the modification was very well. After separation and purification by strong cation exchange column SP-650S, the pure PEG-modified sea anemone neurotoxin rhk2a accounted for 83.287%. For guinea pigs with heart failure, the left ventricular systolic pressure and the maximum change rate of left ventricular systolic pressure fall of the group of mPEG-rhk2a apparently increased and showed a smooth state after 20 min. The positive inotropic effect of mPEG-rhk2a was stronger than the Cedilanid injection but weaker and smoother than the rhk2a injection. CONCLUSION The optimal condition of the modified reaction is successfully found and the cardiac effect of mPEG-rhk2a is very well.