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PubMed2004

Stabilization of immobilized glucose oxidase against thermal inactivation by silanization for biosensor applications.

An important requirement of immobilized enzyme based biosensors is the thermal stability of the enzyme. Studies were carried out to increase thermal stability of glucose oxidase (GOD) for biosensor applications. Immobilization of the enzyme was carried out using glass beads as support and the effect of silane concentration (in the range 1-10%) during the silanization step on the thermal stability of GOD has been investigated. Upon incubation at 70 degrees C for 3h, the activity retention with 1% silane was only 23%, which increased with silane concentration to reach a maximum up to 250% of the initial activity with 4% silane. Above this concentration the activity decreased. The increased stability of the enzyme in the presence of high silane concentrations may be attributed to the increase in the surface hydrophobicity of the support. The decrease in the enzyme stability for silane concentrations above 4% was apparently due to the uneven deposition of the silane layer on the glass bead support. Further work on thermal stability above 70 degrees C was carried out by using 4% silane and it was found that the enzyme was stable up to 75 degrees C with an increased activity of 180% after 3-h incubation. Although silanization has been used for the modification of the supports for immobilization of enzymes, the use of higher concentrations to stabilize immobilized enzymes is being reported for the first time.

Biosensing Techniques↗
PubMed2006

Improving the stability of an antibody variable fragment by a combination of knowledge-based approaches: validation and mechanisms.

Numerous approaches have been described to obtain variable fragments of antibodies (Fv or scFv) that are sufficiently stable for their applications. Here, we combined several knowledge-based methods to increase the stability of pre-existing scFvs by design. Firstly, the consensus sequence approach was used in a non-stringent way to predict a large basic set of potentially stabilizing mutations. These mutations were then prioritized by other methods of design, mainly the formation of additional hydrogen bonds, an increase in the hydrophilicity of solvent exposed residues, and previously described mutations in other antibodies. We validated this combined method with antibody mAbD1.3, directed against lysozyme. Fourteen potentially stabilizing mutations were designed and introduced into scFvD1.3 by site-directed mutagenesis, either individually or in combinations. We characterized the effects of the mutations on the thermodynamic stability of scFvD1.3 by experiments of unfolding with urea, monitored by spectrofluorometry, and tested the additivity of their effects by double-mutant cycles. We also quantified the individual contributions of the resistance to denaturation ([urea](1/2)) and cooperativity of unfolding (m) to the variations of stability and the energy of coupling between mutations by a novel approach. Most mutations (75%) were stabilizing and none was destabilizing. The progressive recombination of the mutations into the same molecule of scFvD1.3 showed that their effects were mostly additive or synergistic, provided a large overall increase in protein stability (9.1 kcal/mol), and resulted in a highly stable scFvD1.3 derivative. The mechanisms of the mutations and of their combinations involved variations in the resistance to denaturation, cooperativity of unfolding, and likely residual structures of the denatured state, which was constrained by two disulfide bonds. This combined method should be applicable to any recombinant antibody fragment, through a single step of mutagenesis.

Animals↗
PubMed2000

Differential regulation of cyclooxygenase-2 (COX-2) mRNA stability by interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) in human in vitro differentiated macrophages.

Cyclooxygenase-2 (COX-2) is a highly inducible gene in macrophages by pro-inflammatory cytokines. A major mechanism for cytokine-induced COX-2 expression is stabilization of COX-2 mRNA. In this study, we examined the induction of COX-2 expression by interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) in human primary in vitro differentiated macrophages. IL-1 beta (5 ng/mL) or TNF-alpha (1 ng/mL) induced up to an approximately 40-fold increase of COX-2 mRNA in macrophages during a 2 to 2.5-hr incubation. Run-off experiments demonstrated that cytokine stimulation had only a mild effect on the COX-2 transcription rate (approximately 10-40% increase). The translation blocker cycloheximide (CHM) (10 mg/mL) superinduced COX-2 mRNA during 2 hr of incubation and further stabilized the COX-2 mRNA (T1/2 > 4 hr). The CHM-superinduced COX-2 mRNA was subject to a rapid degradation after removal of CHM (T1/2 < 1 hr). Both IL-1 beta and TNF-alpha stabilized cytokine-induced COX-2 mRNA (T1/2 > or = 2 hr). Maximal stabilization of COX-2 mRNA after a short-term stimulation required the continued presence of IL-1 beta in the medium. Long-term treatment of TNF-alpha destabilized the induced COX-2 mRNA. Cells simultaneously treated with both IL-1 beta and TNF-alpha had a reduced induction of COX-2, IL-1 beta, and IL-6 mRNA. In transcription-arrested cells, the translation blocker puromycin affected the TNF-alpha-induced stabilization and destabilization of COX-2 mRNA, but not the IL-1 beta-induced stabilization. The studies suggest that positive and negative regulation of mRNA stability may play a major role in cytokine-mediated COX-2 induction in human macrophages. TNF-alpha may play both pro-inflammatory and protective roles during inflammation by regulation of pro-inflammatory gene transcripts.

Cell Differentiation↗
PubMed1999

Bacterial formate dehydrogenase. Increasing the enzyme thermal stability by hydrophobization of alpha-helices.

NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from methylotrophic bacterium Pseudomonas sp.101 exhibits the highest stability among the similar type enzymes studied. To obtain further increase in the thermal stability of FDH we used one of general approaches based on hydrophobization of protein alpha-helices. Five serine residues in positions 131, 160, 168, 184 and 228 were selected for mutagenesis on the basis of (i) comparative studies of nine FDH amino acid sequences from different sources and (ii) with the analysis of the ternary structure of the enzyme from Pseudomonas sp.101. Residues Ser-131 and Ser-160 were replaced by Ala, Val and Leu. Residues Ser-168, Ser-184 and Ser-228 were changed into Ala. Only Ser/Ala mutations in positions 131, 160, 184 and 228 resulted in an increase of the FDH stability. Mutant S168A was 1.7 times less stable than the wild-type FDH. Double mutants S(131,160)A and S(184,228)A and the four-point mutant S(131,160,184,228)A were also prepared and studied. All FDH mutants with a positive stabilization effect had the same kinetic parameters as wild-type enzyme. Depending on the position of the replaced residue, the single point mutation Ser/Ala increased the FDH stability by 5-24%. Combination of mutations shows near additive effect of each mutation to the total FDH stabilization. Four-point mutant S(131,160,184,228)A FDH had 1.5 times higher thermal stability compared to the wild-type enzyme.

Amino Acid Sequence↗
PubMed2002

The effect of salts on the activity and stability of Escherichia coli and Haloferax volcanii dihydrofolate reductases.

The extremely halophilic Archae require near-saturating concentrations of salt in the external environment and in their cytoplasm, potassium being the predominant intracellular cation. The proteins of these organisms have evolved to function in concentrations of salt that inactivate or precipitate homologous proteins from non-halophilic species. It has been proposed that haloadaptation is primarily due to clustering of acidic residues on the surface of the protein, and that these clusters bind networks of hydrated ions. The dihydrofolate reductases from Escherichia coli (ecDHFR) and two DHFR isozymes from Haloferax volcanii (hvDHFR1 and hvDHFR2) have been used as a model system to compare the effect of salts on a mesophilic and halophilic enzyme. The KCl-dependence of the activity and substrate affinity was investigated. ecDHFR is largely inactivated above 1M KCl, with no major effect on substrate affinity. hvDHFR1 and hvDHFR2 unfold at KCl concentrations below approximately 0.5M. Above approximately 1M, the KCl dependence of the hvDHFR activities can be attributed to the effect of salt on substrate affinity. The abilities of NaCl, KCl, and CsCl to enhance the stability to urea denaturation were determined, and similar efficacies of stabilization were observed for all three DHFR variants. The DeltaG degrees (H(2)O) values increased linearly with increasing KCl and CsCl concentrations. The increase of DeltaG degrees (H(2)O) as a function of the smallest cation, NaCl, is slightly curved, suggesting a minor stabilization from cation binding or screening of electrostatic repulsion. At their respective physiological ionic strengths, the DHFR variants exhibit similar stabilities. Salts stabilize ecDHFR and the hvDHFRs by a common mechanism, not a halophile-specific mechanism, such as the binding of hydrated salt networks. The primary mode of salt stabilization of the mesophilic and halophilic DHFRs appears to be through preferential hydration and the Hofmeister effect of salt on the activity and entropy of the aqueous solvent. In support of this conclusion, all three DHFRs are similarly stabilized by the non-ionic cosolute, sucrose.

Amino Acids↗
PubMed2000

Effect of media composition on long-term in vitro stability of barium alginate and polyacrylic acid multilayer microcapsules.

For a number of applications stability of microcapsules is a critical factor. Since the maintenance of polyelectrolyte complexes depends considerably on the ion composition we tested the physical properties of barium alginate capsules and searched for conditions to improve stability by a multilayer coating with polyethylenimine (PEI) and polyacrylic acid (PAA). Mechanical stability and diameters were determined in barium alginate capsules and compared with multilayer capsules. Multilayer coating resulted in smaller capsules than barium complexing alone. The difference was more pronounced when CaCl2 was used instead of NaCl during coating. Barium alginate capsules and application of CaCl2 during coating led to continuous pressure profiles, whereas NaCl resulted in bursting at a defined pressure, indicating the additional contribution to mechanical stability by the outer layers. After 7 d culture, mechanical stability of coated capsules decreased in RPMI and NaCl but was most pronounced in sodium citrate. The capsule diameter increased in sodium citrate, less pronounced in NaCl and was significantly different to RPMI and double distilled water. During long-term culture in RPMI, the diameter increased and mechanical stability decreased significantly. Multilayer coating improved mechanical stability which was impeded most in sodium citrate, to a lesser extent by NaCl and RPMI even after long-term exposure.

Acrylic Resins↗
PubMed1981

Effects of manganese and calcium on conformational stability of concanavalin A: a differential scanning calorimetric study.

The effect of degree of saturation of concanavalin A with Mn2+ or Ca2+, or both, on its thermal denaturation was investigated by differential scanning calorimetry. Acid-demetallized concanavalin A was partly or fully remetallized in acetate buffer (pH 5.0) containing 0.4 to 0.5 M NaCl. Under these conditions, native dimeric concanavalin A is highly stable, undergoing heat denaturation at 101 degrees C, with an enthalpy of denaturation of 7.4 cal/g. Removal of metal ions lowered stability considerably; concanavalin A with 0.06 Mn2+/monomer and 0.23 Ca2+/monomer (mol/mol) was denatured at 74 degrees C with an enthalpy of denaturation of 3.2 cal/g. Added Mn2+ stabilized demetallized concanavalin A, but added Ca2+ alone (up to 2 mol/mol monomer) did not. The Ca2+/ concanavalin A ratio influenced stabilization by Mn2+. In the presence of 1 to 2 Mn2+/ monomer and 0.5 or less Ca2+/monomer (mol/mol), stabilized concanavalin A was denatured at 85-88 degrees C and at 94-97 degress C, indicating presence of two stabilized metallo-concanavalin A species. At 1.0 or more mole each of Mn2+ and Ca2+ per monomer, one endotherm was observed at or above 98 degrees C and the enthalpy of denaturation was increased to 5.3 cal/g from less than 3.6 cal/g at lower metal ion/protein ratios. Stabilization was greater with Mn2+ plus Ca2+ than with Mn2+ alone, consistent with intrasubunit cooperativity in metal ion-induced stabilization of concanavalin A.

Calcium↗
PubMed2000

The effect of mutation at valine-45 on the stability and redox potentials of trypsin-cleaved cytochrome b5.

In an attempt to elucidate the determinants of redox potential and protein stability in cytochrome b5, three mutants at a highly conserved residue Val45, which is a member of heme hydrophobic pocket residues have been characterized. The V45Y mutant was designed to introduce a bulkier residue and a hydroxyl group to the heme pocket. The mutants V45H and V45E were constructed to test the effect of positive and negative charge on the stability and redox potential of proteins. The influence of these mutants on the protein stability towards thermal, urea, acid, ethanol and on the redox potential were studied. It is concluded that the decrease of hydrophobic free energy and the larger volume of the tyrosine make the phenylhydroxyl group of tyrosine still sitting inside the hydrophobic pocket, while the side chain of the mutant V45E and V45H shift away from the heme pocket. The redox potentials of mutants V45Y, V45H, V45E and wild-type of cytochrome b5 are -35 mV, 8 mV, -26 mV and -3 mV, respectively. The bigger change of the V45Y on redox potential is due to the close contact between the hydroxyl group and the heme, while the changes of the V45E and V45H result from the alteration of charge density and distribution around the heme. Different relative stability of these mutants towards heat have been observed with the order: WT > V45Y-V45H > V45E being both in the oxidized and reduced state. The relative stability induced by addition of urea decreases in the order: WT > V45Y > V45H > V45E. These results suggest that the difference in the hydrophobic free energy is a major factor contributing to the stability of the Val45 mutants. Also the loose of the helix III in the mutant V45E makes it more unstable. These results indicate that residue Val45 plays an important role in the stability and redox potential of the protein.

Acids↗
PubMed1997

Stability of ranitidine and thiamine in parenteral nutrition solutions.

Our objectives were to ascertain the stability of thiamine HCl (3 mg/L) and ranitidine HCl (150 mg/L) at room and refrigeration temperatures in a central vein formula of parenteral nutrition (PN) solution (containing 6% amino acid, 25% carbohydrate, macro- and microminerals, and multivitamins) and to determine the effect of ranitidine on the stability of thiamine. Stability of thiamine and ranitidine in PN solutions was also compared with PN-salt solutions, which contained no amino acids or carbohydrates, to indirectly ascertain the impact of these macronutrients on the stability of these moieties. High-pressure liquid chromatography (HPLC) methods were developed to measure thiamine and ranitidine in the PN mixture. Stability studies were conducted in triplicate and each sample was assayed in duplicate using newly developed HPLC methods. Refrigeration provided stability for both ranitidine and thiamine for extended periods of time. At room temperature, ranitidine was also shown to be stable for about 188 h; there was, however, significant degradation of thiamine at 24 h with, and without, addition of ranitidine. The time required for 10% of thiamine to degrade was calculated to be 12.9 h for the PN mixture containing multivitamins and ranitidine; 11.1 h for the PN mixture containing multivitamins alone; and 33.4 h for the PN mixture containing only thiamine HCl. This work suggests that the concentration of thiamine in this central vein PN formula, with or without ranitidine, falls below the 90% acceptable stability within 24 h.

Chromatography, High Pressure Liquid↗
PubMed2003

Stability of sufentanil and levobupivacaine solutions and a mixture in a 0.9% sodium chloride infusion stored in polypropylene syringes.

We have evaluated the chemical and microbiological stability of sufentanil citrate, levobupivacaine hydrochloride and a mixture in a 0.9% sodium chloride infusion in order to provide background information on the storage of a sufentanil-levobupivacaine mixture in polypropylene (PP) syringes. Chemical assays were performed by HPLC on days 0, 1, 2, 3, 8, 14, 23, 28 and 30 after storage at 4, 21, and 36 degrees C. Microbiological stability was evaluated under aseptic conditions using a laminar air flow station, with a grade A environment and a B background. The samples taken for microbiological analysis were collected immediately after preparation of the solutions and then after 7, 14, 21 and 28 days storage. At 4 degrees C the sufentanil citrate solution was stable for 23 days. At 21 degrees C the sufentanil citrate solution maintained chemical stability for 3 days, but thereafter the concentration of sufentanil decreased 15% from day 3 to day 8. At 36 degrees C a similar decrease was noticed from day 1 to day 3. On the contrary, the levobupivacaine hydrochloride solution maintained chemical stability for 28 days at 4 and 21 degrees C and for 23 days at 36 degrees C. The sufentanil-levobupivacaine mixture maintained chemical stability for 28 days at 4, 21 and 36 degrees C. The sufentanil and levobupivacaine solutions and the mixture studied maintained microbiological stability for 28 days. According to the chemical and microbiological stability studies, the sufentanil-levobupivacaine mixture in PP syringes could be stored for 28 days at 4 and 21 degrees C.

Anesthetics, Intravenous↗
PubMed1979

Stability of buffalo casein micelles.

Buffalo skim-milk is less heat stable than cow skim-milk. Interchanging ultracentrifugal whey (UCW) and milk diffusate with micellar casein caused significant changes in the heat stability of buffalo casein micelles (BCM) and cow casein micelles (CCM). Buffalo UCW dramatically destabilized CCM, whereas buffalo diffusate with CCM exhibited the highest heat stability. Cow kappa-casein stabilizes alphas-casein against precipitation by Ca better than buffalo kappa-casein. About 90% of alphas-casein could be stabilized by kappa:alphas ratios of 0.20 and 0.231 for cow and buffalo, respectively. Sialic acid release from micellar kappa-casein by rennet was higher than from acid kappa-casein in both buffalo and cow caseins, the release being slower in buffalo. The released macropeptide from buffalo kappa-casein was smaller than that from cow kappa-casein as revealed by Sephadex gel filtration. Sub-units of BCM have less sialic acid (1.57 mg/g) than whole micelles (2.70 mg/g). On rennet action, 47% of bound sialic acid was released from sub-units as against 85% from whole micelles. The sub-micelles are less heat stable than whole micelles. Among ions tested, added Ca reduced heat stability more dramatically in whole micelles, whereas added phosphate improved the stability of micelles and, more strikingly, of sub-micelles. Citrate also improved the heat stability of sub-micelles but not of whole micelles.

Animals↗
PubMed1995

Stability studies on maize leaf phosphoenolpyruvate carboxylase: the effect of salts.

The solution stability of phosphoenolpyruvate carboxylase (PEPC) has been determined in the presence of various salts by temperature-accelerated enzyme inactivation and also by using high-performance size-exclusion chromatography. Kosmotropic (water structuring) anions in the Hofmeister series (HPO(4)2-, citrate3-, SO(4)2-, F-, OAc-) and glutamate stabilized the enzyme most effectively, while Cl- (a borderline Hofmeister anion) and Br- (a chaotropic anion) were destabilizing. The effects of the cations on PEPC stability ranged from relatively inert (Na+, K+) to destabilizing ((CH3)4N+, NH4+, Li+). The observed stabilization of PEPC by specific salts has been interpreted in terms of the positive surface tension increment and the water-structuring effects conferred on the solution by the specific stabilizing reagents. Both these effects enhance hydrophobic interactions of proteins and increase the energy required to enlarge the surface area of the solvent cavity in which the protein resides. The destabilization of PEPC by some salts at a concentration of 0.5 M was associated with the dissociation of the tetrameric enzyme into its dimeric and monomeric forms, a process most probably occurring as a result of ion-peptide dipole binding, which promotes protein-solvent interaction and a subsequent reduction in the free energy of cavity formation. The stabilization of enzyme activity by kosmotropic salts depended on the salt concentration with maximum stabilization of PEPC in solution at 52 degrees C observed with 0.6-0.8 M sodium glutamate, 2 M KF, and 2.2 M KOAc. Higher concentrations of these salts resulted in decreased activity. This reduction in activity of PEPC in the presence of high concentrations of kosmotropic salts appears to be associated with irreversible conformational changes of the tetrameric enzyme.

Chromatography, High Pressure Liquid↗
PubMed1993

Stabilization of a protein by guanidinium chloride.

Guanidinium chloride is a commonly used denaturant to unfold native proteins and to determine their Gibbs free energy of stabilization, delta Gstab. Here we show that this denaturant has a dual role for the stability and the folding of the model protein ribonuclease T1. When present at low concentration (0-0.3 M), guanidinium chloride stabilizes the folded protein toward thermal and urea-induced unfolding and decreases the rate of unfolding. At high concentration the function of guanidinium chloride as a denaturant dominates and ribonuclease T1 is cooperatively unfolded. Ribonuclease T1 is also strongly stabilized by other salts, such as NaCl, at low concentrations, and the dependence of the thermal stability on salt concentration is not linear. Such a complex behavior was not found in control experiments with pancreatic ribonuclease A. The stabilization in the presence of low concentrations of guanidinium chloride originates probably from the binding of guanidinium ions to one or a few cation binding sites that exist in native ribonuclease T1. It is not observed when an additional salt, NaCl, is present simultaneously. The favorable interaction of guanidinium chloride with the native protein leads to increased values for delta Gstab, when unfolding transitions induced by guanidinium chloride are analyzed on the basis of the two-state model by the linear extrapolation procedure. The noncoincidence of these delta Gstab values with stability data derived from urea-induced or thermal unfolding transitions does not imply that the two-state model is not appropriate but that the linear extrapolation to zero molar denaturant is incorrect.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme Stability↗
PubMed1992

Arginine residues as stabilizing elements in proteins.

Site-specific substitutions of arginine for lysine in the thermostable D-xylose isomerase (XI) from Actinoplanes missouriensis are shown to impart significant heat stability enhancement in the presence of sugar substrates most probably by interfering with nonenzymatic glycation. The same substitutions are also found to increase heat stability in the absence of any sugar derivatives, where a mechanism based on prevention of glycation can no longer be invoked. This rather conservative substitution is moreover shown to improve thermostability in two other structurally unrelated proteins, human copper, zinc-superoxide dismutase (CuZnSOD) and D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus subtilis. The stabilizing effect of Lys----Arg substitutions is rationalized on the basis of a detailed analysis of the crystal structures of wild-type XI and of engineered variants with Lys----Arg substitution at four distinct locations, residues 253, 309, 319, and 323. Molecular model building analysis of the structures of wild-type and mutant CuZnSOD (K9R) and GAPDH (G281K and G281R) is used to explain the observed stability enhancement in these proteins. In addition to demonstrating that even thermostable proteins can lend themselves to further stability improvement, our findings provide direct evidence that arginine residues are important stabilizing elements in proteins. Moreover, the stabilizing role of electrostatic interactions, particularly between subunits in oligomeric proteins, is documented.

Aldose-Ketose Isomerases↗
PubMed2000

Consequences of cAMP-binding site mutations on the structural stability of the type I regulatory subunit of cAMP-dependent protein kinase.

The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with two tandem cAMP-binding domains, A and B. The importance of cAMP binding on the stability of the R subunit was probed by intrinsic fluorescence and circular dichroism (CD) in the presence and absence of urea. Several mutants were characterized. The site-specific mutants R(R209K) and R(R333K) had defects in cAMP-binding sites A and B, respectively. R(M329W) had an additional tryptophan in domain B. Delta(260-379)R lacked Trp260 and domain B. The most destabilizing mutation was R209K. Both CD and fluorescence experiments carried out in the presence of urea showed a decrease in cooperativity of the unfolding, which also occurred at lower urea concentrations. Unlike native R, R(R209K) was not stabilized by excess cAMP. Additionally, CD revealed significant alterations in the secondary structure of the R209K mutant. Therefore, Arg209 is important not only as a contact site for cAMP binding but also for the intrinsic structural stability of the full-length protein. Introducing the comparable mutation into domain B, R333K, had a smaller effect on the integrity and stability of domain A. Unfolding was still cooperative; the protein was stabilized by excess cAMP, but the unfolding curve was biphasic. The R(M329W) mutant behaved functionally like the native protein. The Delta(260-379)R deletion mutant was not significantly different from wild-type RIalpha in its stability. Consequently, domain B and the interaction between Trp260 and cAMP bound to site A are not critical requirements for the structural stability of the cAPK regulatory subunit.

Binding Sites↗
PubMed2001

Monovalent cation-induced conformational change in glucose oxidase leading to stabilization of the enzyme.

Glucose oxidase (GOD) from Aspergillus niger is an acidic dimeric enzyme having a high degree of localization of negative charges on the enzyme surface and dimer interface. We have studied the effect of monovalent cations on the structure and stability of GOD using various optical spectroscopic techniques, limited proteolysis, size exclusion chromatography, differential scanning calorimetry, and enzymic activity measurements. The monovalent cations were found to influence the enzymic activity and tertiary structure of GOD, but no effect on the secondary structure of the enzyme was observed. The monovalent cation-stabilized GOD was found to have a more compact dimeric structure but lower enzymic activity than the native enzyme. The enzyme's K(m) for D-glucose was found to be slightly enhanced for the monovalent cation-stabilized enzyme (maximum enhancement of about 35% for LiCl) as compared to native GOD. Comparative denaturation studies on the native and monovalent cation-stabilized enzyme demonstrated a significant resistance of cation-stabilized GOD to urea (about 50% residual activity at 6.5 M urea) and thermal denaturation (Delta T(m) maximum of 10 degrees C compared to native enzyme). However, pH-induced denaturation showed a destabilization of monovalent cation-stabilized GOD as compared to the native enzyme. The effectiveness of monovalent cations in stabilizing GOD structure against urea and thermal denaturation was found to follow the Hofmeister series: K(+) > Na(+) > Li(+).

Aspergillus niger↗
PubMed1994

Contribution to global protein stabilization of the N-capping box in human growth hormone.

In this work we have investigated the contribution to protein stability of residues forming the boundaries of alpha-helices. At the N-terminus of helix 2 of human growth hormone there are two residues, Ser71 and Glu74, which form two reciprocal hydrogen bonds between the side chains and the backbone nitrogens of either residue (the N-capping box). In order to evaluate the stabilizing effect of each hydrogen bond, site-directed mutagenesis was employed. In addition, the effect of side-chain negative charge on helix stabilization, via charge dipole interaction, was assessed. Ultraviolet spectroscopy and near- and far-UV CD spectroscopy as well as guanidine hydrochloride protein denaturation were used as assays to monitor the conformational and free energy of stabilization changes induced by the point mutations. The results of these experiments can be summarized as follows: (a) receptor binding studies showed that the tertiary conformation of each mutant was similar to that of the native hormone, (b) far-UV CD spectroscopic analyses showed that the overall alpha-helical content was unchanged in the mutants, (c) UV absorption and CD spectroscopic analyses indicated small alterations in helical packing in those mutants in which the hydrogen bond between the side chain of Ser71 and backbone NH of Glu74 was disrupted, (d) the hydrogen bond involving the side chain of Ser71 contributes at least 1.0 kcal/mol to protein stabilization and has a 2-fold larger stabilizing effect than that of the hydrogen bond involving the Glu74 side chain, and (e) the putative charge-dipole interaction of Glu74 with the alpha-helix dipole does not contribute to the stabilization of the tertiary conformation of human growth hormone.

Circular Dichroism↗
PubMed1990

Interactions of serum proteins with small unilamellar liposomes composed of dioleoylphosphatidylethanolamine and oleic acid: high-density lipoprotein, apolipoprotein A1, and amphipathic peptides stabilize liposomes.

Small unilamellar liposomes composed to dioleoylphosphatidylethanolamine (DOPE) and oleic acid (OA) are stabilized by incubation with normal human serum or plasma [Liu, D., & Huang, L. (1989) Biochemistry 28, 7700-7707]. The present report describes a systematic study of interactions of purified serum proteins and lipoproteins with these liposomes. Albumin destabilized liposomes by extracting OA from the liposomes, whereas immunoglobulins and lipoproteins (HDL, LDL, and VLDL) had no effect. However, HDL and, to some extent, VLDL showed a rapid stabilization activity against the lytic effect of albumin. HDL added together with or shortly after the addition of albumin completely abolished the liposome leakage and aggregation effects induced by albumin. SDS-PAGE analysis of the HDL-stabilized liposomes revealed that apolipoprotein A1 was associated with liposomes. Purified apolipoprotein A1, but not a lipid mixture resembling the lipid composition of HDL, showed comparable liposome stabilization activity as HDL. Furthermore, synthetic peptides resembling the amphipathic helices found in apolipoprotein A1 also showed strong liposome stabilization activity. Peptides which were able to form amphipathic helixes of a wedge shape were more effective stabilizers than those which could not. These data indicate that HDL plays a major role in human serum or plasma for the liposome stabilization activity. HDL exerts its activity probably by the interactions of the amphipathic helices of apolipoprotein A1 with the hydrophobic voids found on the outer surface of the highly curved, small liposomes.

Apolipoprotein A-I↗