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AIM: To construct another growth hormone releasing hormone (GHRH) analog, Pro-Pro-hGHRH(1-44)-Gly-Gly-Cys peptide and to compare its activity with that of Pro-Pro-hGHRH(1-44)OH peptide. METHODS: The pro-pro-hGHRH(1-44)-gly-gly-cys DNA fragment was synthesized by polymerase chain reaction. The recombinant protein was expressed to high levels in Escherichia coli BL21 (DE3). The Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys peptide was purified to homogeneity by cell disruption, washing, ethanol precipitation, acid hydrolysis, and SP-Sephadex C-25 and Sephadex G-25 column chromatography. The peptide molecular mass was determined by electrospray ionization mass spectroscopy (ESI-MS). Its purity was determined by SDS-PAGE. The concentration of growth hormone (GH) stimulated by GHRH and its analogs was determined with antiserum kit against human GH. The other human hormones as hTSH, hFSH, hLH, and hPRL were determined with a paramagnetic particle chemiluminescent immunoassay kit. RESULTS: The molecular weight of Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys was 5455.4 kDa which was coincident with the theoretical calculations. All the three peptides at 5 mg/L stimulated GH release from the human fetal pituitary but only the difference between Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys group and blank group was significant (P＜0.05). Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys 0.01, 0.1, and 1 mg/L and Pro-Pro-hGHRH(1-44)OH 0.1 and 1 mg/L stimulated GH release from rat pituitary in a concentration-dependent manner (P＜0.05,P＜0.01). At the same concentration Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys stimulated more GH release than ProPro-hGHRH (1-44)OH. Pro-Pro-hGHRH (1-44)OH 0.01 mg/L and hGHRH (1-40)OH 2 mg/L did not stimulate GH release from rat pituitary (P＞0.05). Pro-Pro-hGHRH (1-44)OH and Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys peptidesat 5 mg/L did not stimulate hTSH, hFSH, hLH, and hPRL release from human fetal pituitary in vitro. CONCLUSION:Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys had a better activity than that of Pro-Pro-hGHRH(1-44)OH. Variation at C-terminus of GHRH could modulate its GH releasing activity.