Derivative UV Absorption Research Articles

Linking the Solution Viscosity of an IgG2 Monoclonal Antibody to Its Structure as a Function of pH and Temperature

Although the viscosity of concentrated antibody solutions has been the focus of many recent studies, less attention has been concentrated on how changes in protein structure impact viscosity. This study examines viscosity profiles of an immunoglobulin G (IgG) 2 monoclonal antibody at 150 mg/mL as a function of temperature and pH. Although the structure of the antibody at pH 4.0-7.0 was comparable at lower temperatures as measured by second derivative UV absorbance and Fourier transform infrared spectroscopy, differences in 8-anilino-1-naphthalene sulfonate (ANS) fluorescence intensity indicated small structural alterations as a function of pH. Below the structural transition onset temperature, the viscosity profiles were pH dependent and linearly correlated with fluorescence intensity, and followed semilogarithmic behavior as a function of temperature. The transitions of the viscosity profiles correlated well with the major structure transitions at a protein concentration of 150 mg/mL. The viscosity correlated particularly well with ANS fluorescence intensity at 0.2 mg/mL below and above the structural transition temperatures. These results suggest: (1) ANS can be an important measure of the overall structure and (2) hydrophobic interactions and charge-charge interactions are the two major physical factors that contribute collectively to the high viscosity of concentrated IgG solutions.

Stability of exenatide in poly(d,l-lactide-co-glycolide) solutions: A simplified investigation on the peptide degradation by the polymer

A solution model can be used to elucidate drug stability issues in a complex system. The aim of this study was to investigate the interaction between poly(d,l-lactide-co-glycolide) (PLGA) and exenatide in organic solvent-acetate buffer saline (ABS) solutions. The effect of solvent composition on exenatide stability was investigated first. In the selected 90:10 dimethyl sulfoxide (DMSO):ABS solution, exenatide stability was examined as a function of PLGA comonomer ratios, molecular weight (Mw) and concentrations. The specific rotation analysis and second derivative UV absorbance spectroscopy were used to monitor the variation of exenatide higher order structure. The effect of ABS pH on the interaction was also investigated. Exenatide degradation products were characterized by HPLC–MS/MS. It was found that exenatide was relatively stable in glacial acetic acid (HAc)–ABS solutions, whereas DMSO content had a strong influence on the conformation state and stability of exenatide. PLGA 50:50 promoted exenatide degradation more than PLGA 75:25 and poly(d,l-lactide) (PLA). Lower Mw and higher concentration of PLGA were beneficial for exenatide degradation. Exenatide was more stable in 90:10 DMSO:ABS (pH 3.0) solution than in 90:10 DMSO:ABS (pH 4.5 and 3.0) solutions during the incubation. HPLC–MS/MS analysis of exenatide demonstrated that acylation was the main degradation route of the peptide.

Physicochemical changes in phosphorylase kinase induced by its cationic activator Mg2+

For over four decades free Mg(2+) ions, that is, those in excess of MgATP, have been reported to affect a wide variety of properties of phosphorylase kinase (PhK), including its affinity for other molecules, proteolysis, chemical crosslinking, phosphorylation, binding to certain monoclonal antibodies, and activity, which is stimulated. Additionally, for over three decades Mg(2+) has been known to act synergistically with Ca(2+) , another divalent activator of PhK, to affect even more properties of the enzyme. During all of this time, however, no study has been performed to determine the overall effects of free Mg(2+) ions on the physical properties of PhK, even though the effects of Ca(2+) ions on PhK's properties are well documented. In this study, changes in the physicochemical properties of PhK induced by Mg(2+) under nonactivating (pH 6.8) and activating (pH 8.2) conditions were investigated by circular dichroism spectroscopy, zeta potential analyses, dynamic light scattering, second derivative UV absorption, negative stain electron microscopy, and differential chemical crosslinking. The effects of the activator Mg(2+) on some of the properties of PhK measured by these techniques were found to be quite different at the two pH values, and displayed both differences and similarities with the effects previously reported to be induced by the activator Ca(2+) (Liu et al., Protein Sci 2008;17:2111-2119). The similarities may reflect the fact that both cations are activators, and foremost among their similarities is the dramatically less negative zeta potential induced by their binding to PhK.

Distinctive structure and interfacial activity of the human apolipoprotein A-IV 347S isoprotein

The T347S polymorphism in the human apolipoprotein (apo) A-IV gene is present at high frequencies among all the world's populations. Carriers of a 347S allele exhibit faster clearance of triglyceride-rich lipoproteins, greater adiposity, and increased risk for developing atherosclerosis, which suggests that this conservative amino acid substitution alters the structure of apo A-IV. Herein we have used spectroscopic and surface chemistry techniques to examine the structure, stability, and interfacial properties of the apo A-IV 347S isoprotein. Circular dichroism spectroscopy revealed that the 347S isoprotein has similar alpha-helical structure but lower thermodynamic stability than the 347T isoprotein. Fluorescence spectroscopy found that the 347S isoprotein exhibits an enhanced tyrosine emission and reduced tyrosine-->tryptophan energy transfer, and second derivative UV absorption spectra noted increased tyrosine exposure, suggesting that the 347S isoprotein adopts a looser tertiary conformation. Surface chemistry studies found that although the 347S isoprotein bound rapidly to the lipid interface, it has a lower interfacial exclusion pressure and lower elastic modulus than the 347T isoprotein. Together, these observations establish that the T347S substitution alters the conformation of apo A-IV and lowers its interfacial activity-changes that could account for the effect of this polymorphism on postprandial lipid metabolism.

Physicochemical changes in phosphorylase kinase associated with its activation

Phosphorylase kinase (PhK) regulates glycogenolysis through its Ca(2+)-dependent phosphorylation and activation of glycogen phosphorylase. The activity of PhK increases dramatically as the pH is raised from 6.8 to 8.2 (denoted as upward arrow pH), but Ca(2+) dependence is retained. Little is known about the structural changes associated with PhK's activation by upward arrow pH and Ca(2+), but activation by both mechanisms is mediated through regulatory subunits of the (alphabetagammadelta)(4) PhK complex. In this study, changes in the structure of PhK induced by upward arrow pH and Ca(2+) were investigated using second derivative UV absorption, synchronous fluorescence, circular dichroism spectroscopy, and zeta potential analyses. The joint effects of Ca(2+) and upward arrow pH on the physicochemical properties of PhK were found to be interdependent, with their effects showing a strong inflection point at pH approximately 7.6. Comparing the properties of the conformers of PhK present under the condition where it would be least active (pH 6.8 - Ca(2+)) versus that where it would be most active (pH 8.2 + Ca(2+)), the joint activation by upward arrow pH and Ca(2+) is characterized by a relatively large increase in the content of sheet structure, a decrease in interactions between helix and sheet structures, and a dramatically less negative electrostatic surface charge. A model is presented that accounts for the interdependent activating effects of upward arrow pH and Ca(2+) in terms of the overall physicochemical properties of the four PhK conformers described herein, and published data corroborating the transitions between these conformers are tabulated.

The response of type three secretion system needle proteins MxiHΔ5, BsaLΔ5, and PrgIΔ5 to temperature and pH

AbstractThe type III secretion system (TTSS) is a specialized supramolecular injectisome composed of 25 or more proteins which form basal and extracellular domains and share gross architectural similarities with bacterial flagella. The extracellular component of the “needle complex” is primarily composed of a single monomeric subunit organized in a helical array surrounding a hollow pore and protrudes from the bacterial membrane. It is through this surface appendage that virulence factors are translocated to the host cell cytoplasm and thereby subvert normal host cell functions. We present here a comprehensive biophysical analysis of the dynamic conformational behavior of the truncated monomeric needle subunit proteins MxiHΔ5 (Shigella flexneri), BsaLΔ5 (Burkholderia pseudomallei), and PrgIΔ5 (Salmonella typhimurium) as well as their thermal stability over a pH range of 3–8. Circular dichroism spectroscopy indicates the secondary structure is largely alpha helical in all three proteins, and surprisingly thermally labile with transition midpoints in the range of 35–50°C over the pH range of 3–8. Additionally, at the concentrations examined, the very broad thermal transitions were >90% reversible. Second derivative UV absorbance spectroscopy data indicates some disruption of the protein's tertiary structure occurs at temperatures in the range of 29–46°C. The difference in the pH of maximal stability for each of the proteins and the variation for each protein with respect to both secondary and tertiary structural elements is striking. It appears, that at physiological temperatures all three proteins experience intermediate non‐native molten globule like states in which they display significant secondary structure in the absence of extensive tertiary interactions. Because of the size difference between the inner pore of the needle and the fully folded needle proteins, it seems clear that the needle subunits must be secreted in a partially or completely unfolded state to reach the distal tip of the needle for assembly. It is proposed that the formation of these intermediate states in the physiological temperature range may play a role in passage through the pore and needle assembly. Proteins 2008. © 2008 Wiley‐Liss, Inc.

Effects of Solutes on Empirical Phase Diagrams of Human Fibroblast Growth Factor 1

A variety of solutes are commonly used to increase the stability of protein in therapeutic formulations. An empirical phase diagram approach is used to evaluate the effects of different types of additives on the solution behavior of a protein of pharmaceutical interest, human fibroblast growth factor 1 (FGF‐1). A specific stabilizer, heparin, and a nonspecific stabilizer, sucrose, were used in this work. The protein was characterized as a function of pH (3–8) and temperature (10–85°C) using Far‐UV circular dichroism (Far‐UV CD), intrinsic and extrinsic fluorescence as well as second derivative UV absorption spectroscopy. Empirical phase diagrams were constructed to summarize the biophysical characterization data obtained with FGF‐1 alone, in the presence of a threefold weight excess of heparin (3× heparin) or 10% sucrose (w/v). Three phases are observed in the low temperature regions at pH 3, 4, and 5–8. Phase boundaries corresponding to major heat‐induced transitions are detected in the physiological temperature range. The highest thermal stabilities are observed near neutral pH (pH 6 and 7). Both heparin and sucrose appear to enhance the thermal stability of FGF‐1, although their effects on the phase diagram are quite distinct. The greatest stabilization is observed at pH 8. Only heparin appears to protect FGF‐1 from acid‐induced unfolding to any extent.

High-throughput screening of stabilizers for Respiratory Syncytial Virus:Identification of stabilizers and their effects on the conformationalthermostability of viral particles

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory infection inchildren worldwide. Recombinant live attenuated viral preparations are one of the most promisingstrategies for vaccination but they typically possess poor thermostability. In this work, a library ofcompounds was screened and stabilizers were selected based on their ability to inhibit theaggregation of RSV perturbed at 56 °C. After screening and selection of excipients, theconformational stability of the RSV proteins was evaluated in the presence of potential stabilizers.The secondary and tertiary structures as well as aggregation/dissociation of RSV were monitoredusing circular dichroism and second derivative UV absorption spectroscopies and light scattering,respectively, as a function of temperature (10-90 °C). RSV membrane fluidity was also evaluatedby generalized polarization of Laurdan fluorescence. Screening experiments showed that a varietyof sugars, amino acids, polyols and polyanions inhibited the aggregation of viral particles.Conformational stability studies demonstrated that the addition of sugars and polyols stabilizedRSV as indicated by a significant increase in the transition melting temperature (Tm) of both thesecondary and tertiary structures as well as the gel to liquid crystalline membrane transition. Theseresults should provide the basis for rational development of more physically stable formulations oflive attenuated RSV vaccines.

Effects of pH and Polyanions on the Thermal Stability of Fibroblast Growth Factor 20

Fibroblast growth factor 20 (FGF20) is a member of the FGF family with potential for use in several different therapeutic categories. In this work, we provide the first structural characterization of FGF20 using a wide variety of approaches. Like other members of the FGF family, FGF20 appears to possess a beta-trefoil structure. The effect of pH on the conformation and thermal stability of FGF20 is evaluated using far-UV circular dichroism (CD), intrinsic and ANS fluorescence, and high-resolution derivative UV absorption spectroscopy. Empirical phase diagrams are constructed to describe the solution behavior of FGF20 over a wide pH and temperature range. The protein appears to be unstable at pH <5, with aggregation and precipitation observed during dialysis. A major heat-induced conformational change also causes aggregation and precipitation of FGF20 at elevated temperatures. The highest thermal stability is observed near neutral pH (Tm ~55 degrees C at pH 7). The effect of several high- and low-molecular mass polyanions on the thermal stability of FGF20 is also examined using CD, intrinsic fluorescence, and DSC analysis. Among these ligands, heparin exhibits the greatest stabilizing effect on FGF20, increasing the Tm by more than 10 degrees C.

Characterization of the interaction between human serum albumin and morin

The interaction of morin with human serum albumin (HSA) has been investigated by using fluorescence, UV absorption and Fourier transform infrared spectroscopic approaches for the first time. Fluorescence data revealed the presence of a specific binding site on HSA for morin, and the binding affinity was 1.13 ± 0.11 × 10 − 5 L Mol − 1 in the physiological condition. The intrinsic fluorescence of morin was conspicuously enhanced in the presence of HSA due to excited-state proton transfer. The binding ability of morin to protein decreased with the increase of the buffer pH from 6.4 to 8.4, which signified that the level of protonation of the hydroxyl groups played an important role during the drug–protein binding process. From the UV absorption spectra of morin in various pH medium, the dissociation behaviors of the hydroxyl groups on the drug molecule were assigned. The second derivative UV absorption spectra of morin after interacting with HSA were used to elucidate the binding mode of morin to protein. The obvious red shift of the UV absorption band I of morin upon binding to HSA further confirmed the formation of HSA–morin complex, and this property was also utilized to estimate the binding constant. The interaction between morin and HSA induced an obvious reduction of the protein α-helix and β-sheet structures.

Conformational lability of two molecular chaperones Hsc70 and gp96: Effects of pH and temperature

Hsc70 and gp96 are two heat shock proteins with molecular chaperone and immune-related activities. The dynamic conformational properties of heat shock proteins appear to play a critical role in their biological activities. In this study, we investigated the effects of pH and temperature on the conformational states of Hsc70 and gp96. The quaternary, tertiary, and secondary structures of both proteins are evaluated by a variety of spectroscopic techniques, including far-UV circular dichroism, Trp fluorescence, ANS fluorescence, and derivative UV absorption spectroscopy. The results are summarized and compared employing an empirical phase diagram approach. Very similar behaviors are seen for both proteins despite their differences in sequence and tertiary structure. Both proteins show substantial conformational lability in responses to the pH and temperature changes of their environment. This study suggests a natural selection for related functional properties through common conformational dynamics rather than immediate structural homology.

Effect of pH and ionic strength on the physical stability of adenovirus type 5

The thermal stability of adenovirus type 5 (Ad5) was investigated over the pH range 3−8 employing a variety of biophysical techniques under conditions of low and high ionic strength. Analysis of the structural stability of Ad5 by dynamic light scattering, intrinsic and extrinsic fluorescence, and second derivative UV absorption spectroscopies suggest that the capsid stability of Ad5 increases with decreasing pH under both ionic strength conditions. Significant aggregation, however, was observed at pH≤5 under conditions of low ionic strength. These studies also suggest that the physical stability of Ad5 is significantly enhanced under acidic conditions in the presence of 1M NaCl. Evaluation of the quaternary structural stability of Ad5 by dynamic light scattering and extrinsic fluorescence spectroscopy suggest that the Ad5 capsid undergoes a two-step dismantling process wherein the viral particles initially expand in size near 50°C and the DNA core is at least partially exposed to the surrounding solvent. Complete capsid disassembly and total exposure of the DNA core follows at higher temperatures. Data generated during these studies were combined employing a multidimensional eigenvector approach that combines data from numerous techniques into a colored representation. This picture, or “empirical phase diagram,” provides an intuitive representation of the physical stability of Ad5 over the pH range 4−8 from 10°C to 85°C. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association

Synergetic effects of pressure and chemical denaturant on protein unfolding: Stability of a serine-type carboxyl protease, kumamolisin

Kumamolisin, a serine carboxyl proteinase, is very stable and hardly denatured by single perturbation of a chemical denaturant (urea), pressure (<500 MPa) or temperature (<65 °C). In order to investigate the cooperative effects of these three denaturing agents, DSC, CD, intrinsic fluorescence, and fourth derivative UV absorbance were measured under various conditions. By application of pressure to kumamolisin in 8 M urea solution, substantial red-shift in the center of fluorescence emission spectral mass was observed, and the corresponding blue-shift was observed for two major peaks in fourth derivative UV absorbance, under the similar urea-containing conditions. The denaturation curves were analyzed on the basis of a simple two-state model in order to obtain thermodynamic parameters (Δ V, Δ G, and m values), and the combined effects of denaturing agents are discussed, with the special interest in the large cavity and neighboring Trp residue in kumamolisin.

Solution Behavior of IFN-β-1a: An Empirical Phase Diagram Based Approach

An empirical phase diagram approach has been developed as a practical tool to aid macromolecular preformulation/formulation studies. This method employs an eigenvector based procedure to visualize and interpret complex data sets. Human Inteferon-beta-1a, an important therapeutic protein, was used to further develop the method and test its utility. The protein was characterized in solution as a function of pH (2-8), temperature (10 degrees C-85 degrees C) and ionic strength (I = 0.1 and 1.0) using intrinsic and ANS fluorescence, Far-UV circular dichroism (Far-UV CD), Fourier Transform Infrared spectroscopy (FTIR) and derivative UV absorbance spectroscopies, as well as differential scanning calorimetry (DSC) to supplement spectroscopic thermal stability studies. Derivative UV absorbance data were initially used to construct a pH-temperature phase diagram at each ionic strength. Three distinctive phases at I = 0.1 and two major phases at I = 1.0 were identified corresponding to different conformation/aggregation states of the protein. For the first time, heterogeneous data sets (i.e., data from different techniques) including Far-UV CD, fluorescence and UV absorbance results were used to generate empirical phase diagrams. Results from different data sets are compared; precautions in applying the method and its overall utility are discussed.

Structural Stability of Adenovirus Type 5

Thermally induced structural changes in adenovirus type 5 (Ad) in the presence of either 2 or 10% sucrose were investigated using a variety of biophysical techniques. In solutions containing 2% sucrose, a highly cooperative transition in the structure of the virus was observed at 45 degrees C as detected by tryptophan fluorescence, derivative UV absorption spectroscopy, circular dichroism (CD), and dynamic and static light scattering. This transition resulted in (at least partial) disassembly of the virus and a concomitant increase in the accessibility of the viral DNA to the fluorescent dye, TOTO-1. Capsid disassembly was verified by transmission electron microscopy, which showed ruptured icosahedral vertices near 45 degrees C followed by complete capsid disassembly at higher temperatures. SDS-PAGE of thermally treated Ad suggests that the penton base (protein III) and protein IIIa (located in the peripentonal region) are significantly more labile than other capsid proteins and may be the initial instigators in capsid disassembly. Additional discrete structural transitions were observed in viral proteins using the aforementioned spectroscopic techniques. Thermally induced rearrangements of the condensed DNA at higher temperatures were also detected by the appearance of "psi"-like features in the CD spectra as well as a dramatic decrease in accessibility of DNA to TOTO-1. These transitions corresponded to discrete endothermic events that are also detected by differential scanning calorimetry. By increasing the concentration of sucrose to 10%, secondary and tertiary structural features of adenoviral proteins were significantly stabilized, although loss of quaternary structure at 45 degrees C was still observed.

New triazine spectroscopic reagent for the separation of dl-amino acids by micellar electrokinetic chromatography

An approach to the chiral separation of racemic mixtures of amino acids by means of micellar electrokinetic chromatography after derivatization with a new triazine spectroscopic reagent, 3-(4,6-dichloro-1,3,5-triazinylamino)-7-dimethylamino-2-methylphenazine (DTDP), has been evaluated. It was found that the derivatives of the aliphatic amino acids such as serine, valine and arginine, could produce a strong UV absorption at 282 nm, whose apparent molar absorptivities are of 10 4 M −1 cm −1, and thus the concentration of the amino acids down to 3×10 −7 M can still give a detectable signal ( S/ N=3). β-Cyclodextrin (β-CD) added to the buffer system was used as a chiral selector, and separation conditions were optimized. The presence of an organic modifier (2-propanol) was also a prerequisite for the chiral separation. The best results for the chiral separation of DTDP-amino acids were achieved in a mixed sodium dodecylsulfate–β-CD–borate–2-propanol medium at pH 9.0. Compared to some of the commonly used derivatization methods, the present one offers a relatively stable derivative and strong UV absorption for the spectroscopically inert amino acids, thus enabling amino acids to be separated and detected by CE even with a simpler UV detector.

Second derivative UV absorbance analysis to monitor nitrate-reduction by bacteria in most probable number determinations

Heterotrophic and autotrophic nitrate-reducing bacteria (NRB) play important roles in many environments. These bacteria are often enumerated by most probable number (MPN) methods. Measuring NO 3 − depletion in the MPN cultures is the definitive way to determine the presence of NRB. Media used for MPN determinations of NRB in oil field waters usually contain high Cl − concentrations, matching those in the water samples. Many methods for measuring NO 3 − concentrations, such as ion chromatography (IC), cadmium reduction and ion electrode methods, are adversely affected by high concentrations of Cl − and organic compounds. A second derivative UV absorbance method proved to be a fast and reliable means for measuring NO 3 − depletion in MPN media used for enumerating autotrophic and heterotrophic NRB, without interferences from Cl − or the organic components in the latter medium. The MPN results for heterotrophic NRB determined by the second derivative UV absorbance agreed well with those determined by the production of nitrous oxide, and were often higher than those determined by measuring nitrate depletion by the diphenylamine spot test.

Pressure-exploration of the 33-kDa protein from the spinach photosystem II particle.

The 33-kDa protein isolated from the spinach photosystem II particle is an ideal model to explore high-pressure protein-unfolding. The protein has a very low free energy as previously reported by chemical unfolding studies, suggesting that it must be easy to modulate its unfolding transition by rather mild pressure. Moreover, the protein molecule consists of only one tryptophan residue (Trp241) and eight tyrosine residues, which can be conveniently used to probe the protein conformation and structural changes under pressure using either fluorescence spectroscopy or fourth derivative UV absorbance spectroscopy. The different experimental methods used in the present study indicate that at 20 degrees C and pH 6, the 33-kDa protein shows a reversible two-state unfolding transition from atmospheric pressure to about 180 MPa. This value is much lower than those found for the unfolding of most proteins studied so far. The unfolding transition induces a large red shift of the maximum fluorescence emission of 34 nm (from 316 nm to 350 nm). The change in standard free energy (DeltaGo) and in volume (DeltaV) for the transition at pH 6.0 and 20 degrees C are -14.6 kJ.mol-1 and -120 mL.mol-1, respectively, in which the DeltaGo value is consistent with that obtained by chemical denaturation. We found that pressure-induced protein unfolding is promoted by elevated temperatures, which seem largely attributed to the decrease in the absolute value of DeltaGo (only a minor variation was observed for the DeltaV value). However, the promotion of the unfolding by alkaline pH seems mainly related to the increase in DeltaV without any significant changes in DeltaGo. It was also found that NaCl significantly protects the protein from pressure-induced unfolding. In the presence of 1 M NaCl, the pressure needed to induce the half-unfold of the protein is shifted to a higher value (shift of 75 MPa) in comparison with that observed without NaCl. Interestingly, in the presence of NaCl, the value of DeltaV is significantly reduced whilst that of DeltaGo remains as before. The unfolding-refolding kinetics of the protein has also been studied by pressure-jump, in which it was revealed that both reactions are a two-state transition process with a relatively slow relaxation time of about 102 s.

Quantitative analysis of chlorpheniramine maleate and phenylephrine hydrochloride in nasal drops by differential-derivative spectrophotometric, zero-crossing first derivative UV spectrophotometric and absorbance ratio methods

Three simple, rapid and accurate methods are described for the simultaneous determination of chlorpheniramine maleate and phenylephrine hydrochloride in two component mixtures. The first method comprised of measurement of difference absorptivities derivatized in first order of a nasal drops in 0.1 N NaOH relative to that of an equimolar solution in methanol at wavelengths of 271.6 and 250.2 nm, respectively. The second method, zero-crossing derivative spectrophotometry, is based on recording the first derivative curves and determining each component using the zero-crossing technique. Using first derivative spectrophotometry, the amplitudes in the first derivative spectra at 246.5 and 238.6 nm were selected to simultaneously determine chlorpheniramine maleate and phenylephrine hydrochloride in the mixture. The presence of identical zero-crossing points for pure drugs and nasal drop solutions established the non-interference of the excipients in the absorption at these wavelengths. Absorbance ratio method was also developed for a comparison method. The proposed procedures were successfully applied to the determination of chlorpheniramine maleate and phenylephrine hydrochloride in nasal drops, with a high percentage of recovery, good accuracy and precision.

Pressure versus heat-induced unfolding of ribonuclease A: the case of hydrophobic interactions within a chain-folding initiation site.

To investigate the characteristics of the postulated carboxy terminal chain-folding initiation site in bovine pancreatic ribonuclease A (RNase A) (residues 106-118), important in the early stages of the folding pathway, we have engineered by site-directed mutagenesis a set of 14 predominantly conservative hydrophobic variants of the protein. The stability of each variant has been compared by pressure and temperature-induced unfolding, monitored by fourth derivative UV absorbance spectroscopy. Apparently simple two-state, reversible unfolding transitions are observed, suggesting that the disruption of tertiary structure of each protein at high pressure or temperature is strongly cooperative. Within the limits of the technique, we are unable to detect significant differences between the two processes of denaturation. Both steady-state kinetic parameters for the enzyme reaction and UV CD spectra of each RNase A variant indicate that truncation of hydrophobic side chains in this region has, in general, little or no effect on the native structure and function of the enzyme. Furthermore, the decreases in free energy of unfolding upon pressure and thermal denaturation of all the variants, particularly those modified at residues 106 and 108, suggest that the hydrophobic residues and side chain packing interactions of this region play an important role in maintaining the conformational stability of RNase A. We also demonstrate the potential of Tyr115 replacement by Trp as a non-destabilizing fluorescence probe of conformational changes local to the region.