Thermodynamic characterization of an intermediate state of human growth hormone

Abstract

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...

Increased stability of human growth hormone with reduced lactogenic potency

“…2). Similar results were obtained in [14] at acid pH. This is indicative of preservation of a significant part of secondary structure in the denatured protein and supports calorimetric data also pointing to a partial unfolding of hGH during heat denaturation due to aggregation.…”

“…3, curve 1) is 48 kcal/mol that is more than two times lower than mean values of denaturation enthalpy corresponding to full unfolding of the near-size proteins [9]. A similar result was obtained for hGH in the acid pH interval [14]. The midpoint of the hGH thermal denaturation was equal to 81 ‡C.…”

Effects of Hydrophilic Cyclodextrins on Aggregation of Recombinant Human Growth Hormone

“…Thus, the ⌬H v / ⌬H c ratio of > 1.0 suggested that the heat-induced disruption of native structure leads to aggregation of rhGH. These results agree with the previous report that above pH 3.5 the thermal denaturation is irreversible due to the aggregation of rhGH upon unfolding (21). The hydrophilic CyDs increased the unfolding temperature (Tm) of rhGH.…”

Protein Quantity on the Air–Solid Interface Determines Degradation Rates of Human Growth Hormone in Lyophilized Samples

“…37 Aggregation of proteins after lyophilization and reconstitution is often correlated with the extent of loss of native protein structure that occurs during the lyophilization process, with those formulations and conditions that yield the greatest loss of native structure resulting in the most aggregation upon reconstitution. 8 Although numerous studies [38][39][40][41][42][43][44][45][46][47] have demonstrated the role of unfolded or partially unfolded protein molecules in fostering aggregation, the acute loss of structure during lyophilization does not completely explain why aggregation levels increases during storage. In current work, by separating the degradation kinetics between surface and bulk of the solid, we find an alternative explanation for protein aggregation during storage.…”