Ultraviolet resonance Raman and fluorescence studies of acid-induced structural alterations in porcine, bovine, and human growth hormone

Kauffman, Emma; Thamann, Thomas J.; Havel, Henry A.

doi:10.1021/ja00196a059

Cited by 21 publications

(13 citation statements)

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“…We note a significant increase in the fluorescence intensity with λ max = 343 nm for photolyzed rbST compared with λ max = 342 nm for native rbST. Such an increase in fluorescence intensity indicates a significant loss of quenching of the Trp fluorescence, through a change in Trp environment and possibly through a modified interaction with the protein disulfide bridges 18–21. However, the minimal 1‐nm shift of the emission maximum suggests no significant modification of the hydrophilicity/hydrophobicity around Trp.…”

Section: Resultsmentioning

confidence: 99%

Solid-State Photodegradation of Bovine Somatotropin (Bovine Growth Hormone): Evidence for Tryptophan-Mediated Photooxidation of Disulfide Bonds

Miller

Hageman²,

Thamann³

et al. 2003

Journal of Pharmaceutical Sciences

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Section: Resultsmentioning

confidence: 99%

Solid-State Photodegradation of Bovine Somatotropin (Bovine Growth Hormone): Evidence for Tryptophan-Mediated Photooxidation of Disulfide Bonds

Miller

Hageman²,

Thamann³

et al. 2003

Journal of Pharmaceutical Sciences

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“…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).…”

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confidence: 99%

Thermodynamic characterization of an intermediate state of human growth hormone

et al. 1998

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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...

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“…Fluorescence intensity is shown in arbitrary units adjusted for the contribution of the Raman spectra of the buffer. The quantum yield of the Met(l-190)PGH 22 kDa form was 0.132 relative to the quantum yield of A'-acetyltryptophanamide (NATA) at pH 8 of 0.14 (Kauffman et al, 1989) with a maximum emission at 336 nm.…”

Section: Resultsmentioning

confidence: 96%

“…The quantum yields of the Met(l-190)PGH 22 kDa form and of the truncated recombinant product, P-band, were recorded as 0.132 and 0.077, respectively, relative to the quantum yield of iV-acetyltryptophanamide (NATA) at pH 8 of 0.14 (Kauffman et al, 1989), and fluorescence emissions were maximum at 336 and 333 nm, respectively (Puri et al, 1993). Whereas the quantum yield of intrinsic fluorescence of Met(l-190)PGH and P-band is dissimilar (Puri et al, 1993), the urea denaturation of PGH and of P-band follow a very similar relationship with urea concentration, with the greatest degree of denaturation occurring between 5 and 9 M urea having midpoints at approximately 7.1 M and 6.8 M urea for Met(l-190)PGH and P-band, respectively (Figure 8, Table 2).…”

Section: Resultsmentioning

confidence: 99%

Comparing the Refolding and Reoxidation of Recombinant Porcine Growth Hormone from a Urea Denatured State and from Escherichia coli Inclusion Bodies

Cardamone¹,

Puri²,

Brandon³

1995

Biochemistry

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Overexpression of cloned genes in bacteria often leads to insoluble refractile body formation requiring solubilization and refolding to obtain biologically active proteins. A refolding pathway was established for a model protein, porcine growth hormone (PGH), yielding an appreciably high recovery of 85%. The conditions include the dilution of a urea, beta-mercaptoethanol (beta-ME) denatured PGH solution in a refolding environment containing 3.5 M urea and 10 mM beta-ME/HED at a 10:1 ratio at pH 9.1 and 0.5 mg/mL PGH. The intrinsic fluorescence-detected transition of PGH in urea gives 3.8 kcal/mol for the free energy of denaturation (delta GH2O) of PGH. The native-like conformation of PGH is dependent on disulfide bonds because reduced and carboxymethylated PGH is devoid of tertiary structure as assessed by intrinsic tryptophan fluorescence. Physical analysis of C-terminally truncated recombinant PGH indicated no significant difference in the free energy of denaturation of P-band in urea as full-length PGH. This suggests that the first disulfide, forming the large loop domain of PGH, provides a significantly greater contribution to the conformational stability of PGH than the second disulfide, which forms the carboxy-terminal small loop domain. The rate of formation of native structure during refolding was biphasic, with native structure identified by intrinsic fluorescence and hydrophobicity spectroscopy prior to disulfide bond formation. Thus "framework" intermediates are prerequisites for correct disulfide formation and tertiary folding of PGH. This study shows how a protein refolds, forms disulfides, and self-associates, which may be useful for examining the refolding of other recombinant proteins.

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Ultraviolet resonance Raman and fluorescence studies of acid-induced structural alterations in porcine, bovine, and human growth hormone

Cited by 21 publications

References 5 publications

Solid-State Photodegradation of Bovine Somatotropin (Bovine Growth Hormone): Evidence for Tryptophan-Mediated Photooxidation of Disulfide Bonds

Solid-State Photodegradation of Bovine Somatotropin (Bovine Growth Hormone): Evidence for Tryptophan-Mediated Photooxidation of Disulfide Bonds

Thermodynamic characterization of an intermediate state of human growth hormone

Comparing the Refolding and Reoxidation of Recombinant Porcine Growth Hormone from a Urea Denatured State and from Escherichia coli Inclusion Bodies

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