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  • 11
    Electronic Resource
    Electronic Resource
    Protein stabilization and destabilization by guanidinium salts (1984)
    s.l. : American Chemical Society
    Biochemistry 23 (1984), S. 5924-5929 
    Details
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1021/bi00320a005
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  • 12
    Electronic Resource
    Electronic Resource
    Densimetric determination of equilibrium binding of sucrose octasulfate with basic fibroblast growth factor (1993)
    Springer
    The protein journal 12 (1993), S. 689-693 
    Details
    ISSN: 1573-4943
    Keywords: Sucrose octasulfate ; equilibrium binding ; densimetry ; basic FGF
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Fibroblast growth factors (FGFs) strongly bind to heparin and are thereby stabilized against deactivation and proteolytic cleavage. Sucrose octasulfate (SOS), which has a chemical structure resembling the repeating unit of heparin, has also been shown to enhance stability of basic FGF against thermal denaturation and to induce a small conformational change. We have examined SOS binding to bFGF using equilibrium dialysis. The difference in SOS concentration across the dialysis membrane was measured using a precision density meter, since the density of SOS differs greatly from that of water. With care, this densimetric technique can measure binding with a precision of ± 0.1 mol/mol using about 2 mg/ml of protein. These results show that the binding saturates at 2 mol of SOS per mole of bFGF as the SOS concentration increases to 3.6 mM or higher. The effect of SOS on the thermal stability of bFGF was examined using denaturation at a constant heating rate, by both turbidity and differential scanning calorimetry. Since the thermal denaturation is irreversible, the temperature where aggregation abruptly increases was taken to indicate the onset of denaturation. This temperature increased by ∼12°C as the SOS concentration increased from 0.018 to 3.6 mM and remained constant above 3.6 mM, consistent with our binding data if the binding is specific to the native state.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01024927
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  • 13
    Electronic Resource
    Electronic Resource
    Stability of fungal α-amylase in sodium dodecylsulfate (1992)
    Springer
    The protein journal 11 (1992), S. 111-117 
    Details
    ISSN: 1573-4943
    Keywords: Unfolding of α-amylase ; SDS resistance of α-amylase ; melting of SDS-amylase complex ; SDS-PAGE
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Unfolding of a fungal α-amylase in aqueous sodium dodecylsulfate (SDS) solution was examined by SDS-polyacrylamide gel electrophoresis (PAGE). When the α-amylase was incubated with 1% SDS at room temperature and subjected to SDS-PAGE, it showed a much higher mobility than expected from the molecular weight. Circular dichroic and gel filtration analyses indicated that the protein is apparently in the native conformation upon incubation with 1% SDS. When the protein was heated in the presence of 1% SDS at 90°C for 10 min, it had a lower mobility in SDS-PAGE and showed characteristics of an unfolded protein by circular dichroism and gel filtration. The melting temperatures of the protein were determined in the absence and presence of SDS by incubating it for 10 min at various temperatures. The melting temperatures were 70, 55, and 49°C in the presence of 0, 1, and 2% SDS, respectively. The observed small shift of the melting temperatures by SDS suggests that the destabilizing action of SDS on the α-amylase is weak. However, the unfolding in SDS is not reversible process, since prolonged incubation of the protein with 1% SDS at 50°C gradually increased the amount of unfolded protein. This indicates that the SDS-induced unfolding of the α-amylase is a slow process.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01025216
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  • 14
    Electronic Resource
    Electronic Resource
    Induced resistance of trypsin to sodium dodecylsulfate upon complex formation with trypsin inhibitor (1992)
    Springer
    The protein journal 11 (1992), S. 171-176 
    Details
    ISSN: 1573-4943
    Keywords: SDS resistance ; trypsin-trypsin inhibitor complex ; SDS-PAGE of trypsin ; melting of SDS-tryspin-inhibitor complex
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The stabilities of trypsin and soybean trypsin inhibitor in sodium dodecylsulfate (SDS) were examined by SDS-polyacrylamide gel electrophoresis (PAGE). Both samples contained several bands, all of which migrated to positions corresponding to the appropriate molecular weight or less, even when the samples were unheated, suggesting that both the trypsin and trypsin inhibitor are susceptible to SDS-induced denaturation. When they were mixed together prior to addition of SDS-PAGE sample buffer (1% SDS), a new smearing band appeared which corresponded to a molecular weight of around 46,000, suggesting that these proteins form a stable complex in SDS. This was confirmed by electroblotting and sequence analysis, which indicated that this band contains both the trypsin and inhibitor sequences. At a fixed concentration of the inhibitor, increasing concentrations of the trypsin resulted in an increase in the intensity of the complex band. When the mixture was heated for 10 min in 1% SDS, the complex band disappeared in a temperature-dependent manner. The melting temperature determined under the experimental conditions used was about 35|MoC. Similar results were obtained with Bowman-Birk trypsin inhibitor, except that the complex with the above inhibitor had a higher melting temperature, around 41|MoC, suggesting that the Bowman-Birk inhibitor/trypsin complex is more stable than the soybean inhibitor/trypsin complex.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01025222
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  • 15
    Electronic Resource
    Electronic Resource
    Characterization of two fluorescent tryptophans in recombinant human granulocyte-colony stimulating factor: Comparison of native sequence protein and tryptophan-deficient mutants (1993)
    Springer
    The protein journal 12 (1993), S. 229-236 
    Details
    ISSN: 1573-4943
    Keywords: Granulocyte-colony stimulating factor ; fluorescent tryptophans ; mutagenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract In order to probe the role of the individual tryptophans of granulocyte-colony stimulating factor (G-CSF) inpH and guanidine HCl-induced fluorescence changes, site-directed mutagenesis was used to generate mutants replacing Trp118, Trp58, or both with phenylalanine. Neither Trp to Phe mutation affected the folding or activity of the recombinant G-CSF, and the material expressed in yeast behaved identically to that expressed inEscherichia coli. All of the G-CSF species responded topH and guanidine HCl in qualitatively the same manner. Trp58 has a fluorescence maximum at 350 nm and is quenched to a greater extent by the addition of guanidine HCl, indicating that it is fully solvent-exposed. Trp118 has a fluorescence maximum at 344 nm, and is less solvent-accessible than Trp58. The analog in which both tryptophans have been replaced with phenylalanine shows only tyrosine fluorescence, with a peak at 304 nm which decreases with increasingpH. The intensity of the tyrosine fluorescence in this analog is much greater than that of the native sequence protein or single tryptophan mutants, indicating that energy transfer is taking place from tyrosine to tryptophan in these molecules. Below neutralpH the tyrosine fluorescence is much greater in the [Phe58]G-CSF than in the [Phe118]G-CSF, indicating that Trp58 might be a more efficient recipient of energy transfer from the tyrosine(s).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01026045
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  • 16
    Electronic Resource
    Electronic Resource
    Cysteine 17 of recombinant human granulocyte-colony stimulating factor is partially solvent-exposed (1993)
    Springer
    The protein journal 12 (1993), S. 525-531 
    Details
    ISSN: 1573-4943
    Keywords: Circular dichroism ; FTIR ; disulfide exchange ; G-CSF ; sulfhydryl titration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Oh-edaet al. have shown instability of granulocyte-colony stimulating factor (G-CSF) upon storage abovepH 7.0 [J. Biol. Chem. (1990)265, 11,432–11,435]. To clarify the mechanism of this instability, the accessibility of a free cysteinyl residue at position 17 for disulfide exchange reaction was examined using a sulfhydryl reagent. The results show that the cysteine is partially solvent-exposed in both glycosylated and nonglycosylated forms, suggesting that the exposure of the cysteine plays a critical role in the instability of the protein. This is supported by the facts that at lowpH where the cysteine is protonated, both proteins have much greater stability and that a Cys17 → Ser analog is extremely stable at neutralpH and 37°C. It was observed that the rate of sulfhydryl titration is slower for the glycosylated form than for the nonglycosylated form, suggesting that the cysteine residue is less solvent-exposed for the former protein or that the pK a is somewhat more basic. In either case, the carbohydrate appears to affect the reactivity of the sulfhydryl group through steric hindrance or alteration in local conformation. Both the glycosylated and nonglycosylated proteins showed essentially identical conformation as determined by circular dichroism, fluorescence, and infrared spectroscopy. Unfolding of these two proteins, induced either by guanidine hydrochloride or bypH, showed an identical course, indicating comparable conformational stability. Contribution of conformational changes to the observed instability at higherpH is unlikely, since little difference in fluorescence spectrum occurs betweenpH 6.0 and 8.0. Based on these observations, G-CSF, whether glycosylated or not, should not be stored above pH 7.0 in solution. On the other hand, G-CSF is extremely stable in acidic solution as expected from the proposed mechanism.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01025117
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  • 17
    Electronic Resource
    Electronic Resource
    Yeast-derived recombinant human insulin-like growth factor I: Production, purification, and structural characterization (1990)
    Springer
    The protein journal 9 (1990), S. 95-104 
    Details
    ISSN: 1573-4943
    Keywords: Insulin-like growth factor ; glycosylation ; disulfide pairing ; circular dichroism ; mutation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Recombinant human insulin-like growth factor I (IGF-I) is efficiently expressed and secreted fromSaccharomyces cerevisiae using a yeast α-factor leader to direct secretion. However, approximately 10–20% of the IGF-I was in a monomeric form, the remaining materials being disulfide-linked aggregates. When the purified material was subjected to reverse-phase high-performance liquid chromatography (rp-HPLC), it gave two doublet peaks, I and II. Upon reduction, doublet peaks I and II converged to one doublet peak. This suggests that peaks I and II result from different disulfide structures, and the doublet feature of each peak results from other causes. Different disulfide structures between peaks I and II were also suggested from the near UV circular dichroism of these proteins. Only the peak II was biologically active, indicating that peak II has the correct disulfide structure. Concanavalin A affinity chromatography of the purified peak II doublet showed binding of the subpeak with an earlier rp-HPLC retention time, indicating that it was glycosylated. Sequence analysis of tryptic peptides suggested that Thr29 was the site of glycosylation. Site-directed mutagenesis was used to convert Thr29 to Asn29. This substitution reduced, but did not eliminate IGF-I glycosylation, suggesting additional glycosylation sites. The site of carbohydrate addition was consistent with the model that O-glycosylations occur on hydroxyl amino acids near proline residues in β-turns.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01024990
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  • 18
    Electronic Resource
    Electronic Resource
    Mutation of Arg55/56 to Leu55/Ala56 in insulin-like growth factor-I results in two forms different in disulfide structure and native conformation but similar under reverse-phase conditions (1993)
    Springer
    The protein journal 12 (1993), S. 247-254 
    Details
    ISSN: 1573-4943
    Keywords: IGF refolding ; IGF analogs ; peptide map ; hydrophobic interaction chromatography ; disulfide structure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Folding of recombinant human insulin-like growth factor-I (IGF-I) results in two distinct species as resolved by reversed-phase high-performance liquid chromatography (RP-HPLC). The earlier eluting peak (PI) has a nonnative disulfide structure, while the later eluting peak (PII) assumes the native disulfide structure. This folding problem causes a lower yield and requires expensive RP-HPLC separation. In contrast, IGF-II folds mainly into a single form with all three disulfide bonds correctly formed. Sequence comparison of the two molecules revealed that IGF-I has arginine at residues 55 and 56, while IGF-II has alanine and leucine, respectively, at these positions. Two analogs of IGF-I, IGF-I (Ala55/Leu56) and IGF-I (Leu56), behave similarly to IGF-II upon refolding and RP-HPLC; that is, a single peak eluted from the RP-HPLC column. However, when the peaks isolated by RP-HPLC were subjected to hydrophobic interaction chromatography, circular dichroism, and peptide mapping, they were found to be a mixture of PI and PII. It was then concluded that factors other than just these two residues contribute to correct folding of IGF-II and that the PI and PII of the above two IGF-I mutants assume different conformation at neutralpH but similar conformation under the RP-HPLC condition.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01028187
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  • 19
    Electronic Resource
    Electronic Resource
    Aggregation Pathway of Recombinant Human Keratinocyte Growth Factor and Its Stabilization (1994)
    Springer
    Pharmaceutical research 11 (1994), S. 1581-1587 
    Details
    ISSN: 1573-904X
    Keywords: Keratinocyte Growth Factor ; Aggregation Pathway ; Protein Formulation ; Protein Stability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Recombinant human keratinocyte growth factor (rhKGF) is prone to aggregation at elevated temperatures. Its aggregation pathway is proposed to proceed initially with a conformational change which perhaps results from repulsion between positively charged residues in clusters forming heparin binding sites. Unfolding of the protein leads to formation of large soluble aggregates. These soluble aggregates then form disulfide cross-linked precipitates. Finally these precipitates are converted to scrambled disulfides and/or non-disulfide cross-linked precipitates. Stabilizers such as heparin, sulfated polysaccharides, anionic polymers and citrate can greatly decrease the rate of aggregation of rhKGF at elevated temperatures. These molecules may all act by reducing charge repulsion on the protein thus stabilizing the native conformation. EDTA, on the other hand, is found to inhibit disulfide formation in aggregates and has only a moderate stabilizing effect on rhKGF.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018905720139
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  • 20
    Electronic Resource
    Electronic Resource
    Conformational changes of recombinant human granulocyte-colony stimulating factor induced bypH and guanidine hydrochloride (1991)
    Springer
    The protein journal 10 (1991), S. 359-367 
    Details
    ISSN: 1573-4943
    Keywords: Granulocyte-colony stimulating factor ; conformational changes ; circular dichroism ; guanidine-induced denaturation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Fluorescence and circular dichroism were used to follow thepH-dependent conformational changes of granulocyte colony stimulating factor (G-CSF). Tryptophan fluorescence of the spectra monitored at 344 nm, or after deconvolution of the emission spectra, at 345 nm, showed a decrease in intensity on going frompH 7 to 4, with a midtransitionpH of 5.8. On the other hand, tyrosine fluorescence measured either by the ratio of intensity at 308 nm to that at 344 nm, or by the fluorescence intensity at 303 nm after deconvolution of the spectra, increased in intensity as thepH was changed from 6 to 2.5, with a midtransitionpH of 4.5. Near UV circular dichroic spectra also showed changes betweenpH 7.5 and 4.5, which correlated with the transition monitored by the tryptophan fluorescence. The guanidine hydrochloride-induced conformational changes of G-CSF at fivepH values from 2.5 to 7.5 were also studied. Circular dichroic and fluorescence spectra revealed minor conformational changes by the addition of 1 or 2 M guanidine HCl at allpH values examined, while the major conformational transition occurred between 2 and 4 M guanidine hydrochloride. The secondary structure of the protein was most stable betweenpH 3.3 and 4.5. The guanidine HCl-induced denaturation of G-CSF involved more than a two-state transition, with detectable intermediate(s) present, and the structure of the intermediate(s) appeared to depend on thepH used. These results are consistent with thepH dependence of the structure described above, and demonstrate the complex conformational properties of G-CSF.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01025250
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