The thermal denaturation of recombinant human growth hormone (rhGH) was studied by differential scanning calorimetry and circular dichroism spectroscopy (CD). The thermal unfolding is reversible only below pH 3.5, and under these conditions a single two-state transition was observed between 0 and 100°C. The magnitudes of the A H and ACp of this transition indicate that it corresponds to a partial unfolding of rhGH. This is also supported by CD data, which show that significant secondary structure remains after the unfolding. Above pH 3.5 the thermal denaturation is irreversible due to the aggregation of rhGH upon unfolding. This aggregation is prevented in aqueous solutions of alcohols such as n-propanol, 2-propanol, or 1,2-propanediol (propylene glycol), which suggests that the self-association of rhGH is caused by hydrophobic interactions. In addition, it was found that the native state of rhGH is stable in relatively high concentrations of propylene glycol (up to 45% v/v at pH 7-8 or 30% at pH 3) and that under these conditions the thermal unfolding is cooperative and corresponds to a transition from the native state to a partially folded state, as observed at acidic pH in the absence of alcohols. In higher concentrations of propylene glycol, the tertiary structure of rhGH is disrupted and the cooperativity of the unfolding decreases. Moreover, the CD and DSC data indicate that a partially folded intermediate with essentially native secondary structure and disordered tertiary structure becomes significantly populated in 70-80% propylene glycol.Keywords: circular dichroism; differential scanning calorimetry; folding intermediates; human growth hormone; propylene glycol; protein folding Recombinant human growth hormone (rhGH) is a single-chain protein of 191 amino acids. Its tertiary structure is characterized by a four-helix bundle with two long loops (de Vos et al., 1992;Ultsch et al., 1994). The native state is stable and does not undergo significant conformational changes between pH 2-1 1 (Tumer et al., 1983;Kauffman et al., 1989; Abildgaard et al., 1992). Slight differences observed in the near-UV CD spectra below pH 4 have been shown to stem from local adjustments in the tertiary structure after the protonation of carboxyl groups, and not from major tertiary structure alterations (Kauffman et al., 1989;DeFelippis et al., 1995).The guanidine hydrochloride (GuHCI) denaturation of rhGH has been extensively studied (Brems et al., 1990;DeFelippis et al., 1993DeFelippis et al., , 1995 Bam et al., 1996). rhGH unfolds at about 4.5 M Reprint requests to: Isabel Gomez-Orellana, Emisphere Technologies, Inc., 15 Skyline Drive, Hawthorne, New York 10532; e-mail: igomez@ emisphere.com. Abbreviations: CD, circular dichroism; AC,, heat capacity change; AG, Gihbs free energy change; AH, enthalpy change; AH,,,, enthalpy change at the unfolding temperature; DSC, differential scanning calorimetry; T,,,, temperature of the maximum in the heat capacity function; rhGH, recombinant human growth hormone; AHUh/AH,,,, van...