pH Of Maximum Stability Research Articles

FORCED DEGRADATION AND PH STABILITY STUDIES OF LYSERGOL: AN ERGOLIC ALKALOID BY STABILITY INDICATING RP HPLC-UV METHOD

A simple HPLC-UV method was developed and validated for the analysis of lysergol, its stress degradation profile, and pH stability studies. Instrument used was Agilent HPLC 1120 autosampler with a C18 reversed phase column and a mobile phase comprised of methanol and water (1% acetic acid) with isocratic flow. Flow rate was kept constant at 1 mL/min throughout the experiment and elution was monitored at 225 nm using UV detector. The calibration plot showed good linear relationship with r 2 = 0.9997. A well resolved single peak was observed at retention time 2.577 minute with asymmetry <1.0. The limit of detection (1.3 µg/mL), limit of quantitation (4.0 µg/mL), RSD (%) (<2.0) and capacity factor (k' = 1.577) were found to be within the acceptable limits. The stress degradation studies revealed lysergol recovery of 99.0% (wet heat induced), 65.9% (base induced), 63.7% (acid induced), 69.7% (H 2 O 2 induced), and 41.7% (UV light induced); whereas, complete drug degradation occurred at dry heat and photochemical induced degradation. From the pH rate profile studies, pH of maximum stability for lysergol was found to be 2.0.

The solution, solid state stability and kinetic investigation in degradation studies of lercanidipine: study of excipients compatibility of lercanidipine

The objectives of this research were to evaluate the stability of lercanidipine in solution state and solid state and explore the compatibility of drug with oils, surfactants and cosurfactants as excipients. The effect of pH on the degradation in solution state was studied through pH-rate profile of lercanidipine in constant ionic strength buffer solutions in pH range 1–8 which gives the pH of maximum stability. Powdered lercanidipine was stored under 40°C/0%~75% relative humidities (RH) or 0% RH/5~50°C to study the influence of RH and temperature on the stability of lercanidipine in solid state. Binary mixtures of lercanidipine and different excipients were stored at 40°C/75% RH, 40°C and at room temperature for excipient compatibility evaluation. The degradation of lercanidipine at different pH appears to fit a typical first-order reaction, but in solid state, it does not fit any obvious reaction model. Moisture content and temperature both play important roles affecting the degradation rate. Lercanidipine exhibits good compatibility with surfactants, cosurfactants and oils as excipients under stressed conditions of different storage temperature in a 3-week screening study. Moreover, the proposed high-performance liquid chromatography method was utilized to investigate the kinetics of the acidic and alkaline degradation processes of lercanidipine at different temperatures.

Development of a vesicular system for effective ocular delivery of acetazolamide A comprehensive approach and successful venture

Acetazolamide (ACZ), a carbonic anhydrase inhibitor (CAI), is a commonly used agent administered orally for the treatment of glaucoma, but many patients cannot tolerate long-term treatment with ACZ because of its systemic side effects. Development of a topically effective formulation of ACZ has not been possible because of its unfavorable partition coefficient, solubility, permeability coefficient, and poor stability at the pH of its maximum solubility. Based on these factors and the ability of niosomes to come into complete contact with corneal and conjunctival surfaces, we used niosomal drug delivery to enhance the corneal absorption of ACZ. Furthermore, the non-ionic surfactants used in the preparation may also temporarily increase the permeability of the corneal epithelium. Earlier we reported reverse-phase evaporation (REV) as the most suitable method for preparing ACZ niosomes. Coating these niosomes with a bioadhesive polymer such as Carbopol 934P (REVbio), which prolongs the contact time of the formulation in the eyes, is another approach reported by us. Boric acid solution (2%), which is isotonic with tears, was used as a vehicle for the developed niosomal formulations because the pH of maximum stability for ACZ is 4.0. By using boric acid solution, pH was maintained between 4-5. We recommend boric acid as the vehicle for ACZ eye drops for the first time. The pharmacodynamic studies showed > 30% fall in intraocular pressure (IOP) which was sustained for up to 5 h. The effect compared well with a four-times higher concentration of dorzolamide (Dorzox), a topical CAI available in the market. Furthermore, microdialysis showed that the ACZ disposition from the REVbio formulation had a peak concentration of 14.94 μg/ml in the aqueous humor, which was almost two times higher than the concentration obtained with the equivalent amount of acetazolamide control suspension. Finally, the developed formulation was evaluated for stability, safety (in terms of its corneal cell toxicity), and the preservative ’ s (sodium perborate) ability to retard contamination (challenge test, BP).

Design of an IV Formulation of an Unstable Prodrug Candidate for Prostate Cancer Treatment: Solution Chemistry of N-(glutaryl-hyp-ala-ser-cyclohexylglycyl-gln-ser-leu)-doxorubicin

ABSTRACTThe chemical degradation of N-(glutaryl-hyp-ala-ser-cyclohexylglycyl-gln-ser-leu)-doxorubicin (henceforth referred to as doxorubicin peptide conjugate 1) was studied in buffered aqueous solution. The pH-rate profile of degradation shows that the doxorubicin conjugate is most stable between pH 5 and 6. The dependence of log kobsd on pH in acidic medium is characteristic of specific acid-catalysis of the sugar hemiaminal of 1 (as in the case of doxorubicin). Isolation of degradates and structural determination shows that the degradation at lower pH values yields the water-insoluble aglycone doxorubicinone, supporting the mechanism of acid-catalyzed loss of the amino sugar. At pH higher than 5, a more complicated degradation pattern is observed, including the loss of the amino sugar and the aromatization of the saturated ring to give 7,8-dehydro-9,10-desacetyldoxorubicinone as one of the major products. Around the pH of maximum stability in solution, the rate of degradation of 1 is significantly greater than that for doxorubicin, which rules out the formulation of a room temperature solution product with a sufficiently long shelflife for market use. Design of a stable lyophilized formulation for sterile reconstitution based on the physicochemical properties of 1 is described.

Kinetics and mechanism of hydrolysis of efavirenz.

To determine the kinetics and mechanism of hydrolysis of efavirenz [(S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one] in aqueous solutions. The solution stability was examined at 60 degrees C and an ionic strength of 0.3 M over the pH range of 0.6 to 12.8. The loss of efavirenz and the appearance of degradants were followed with a reverse-phase high-performance liquid chromatography assay. Characterization of the degradants was accomplished with liquid chromatography-mass spectrometry. The degradation of efavirenz followed apparent first-order kinetics over the pH range of 0.6 to 12.8 at 60 degrees C. The catalytic effect of phosphate and borate buffers was negligible while acetate and citrate demonstrated buffer catalysis. The overall rate constant indicated a pH minimum (the pH of maximum stability) of approximately 4. Mass spectra data identified a degradant with a molecular weight consistent with hydrolysis of the cyclic carbamate to the corresponding amino alcohol. The degradation route was confirmed with spiking experiments with an authentic sample of the amino alcohol indicating that the carbamate hydrolysis pathway was the predominant reaction throughout the pH range studied. Subsequent degradation of the amino alcohol proceeded at the extremes of the pH range studied via rearrangement to the quinoline. The pH-rate profile was consistent with a combination of a V-shaped profile in the pH range of 0-9 and a sigmoid-shaped profile in the pH range of 4-13. The plateau that began at pH 10-11 is a result of the ionization of the amine of the carbamate inhibiting the base-catalyzed hydrolysis of efavirenz, given that the ionized form of the carbamate is resonance-stabilized toward hydroxide-catalyzed degradation. Thus, increasing the pH resulted in a parallel decrease in the unionized fraction and increase in hydroxide ion concentration resulting in a constant k(obs) value.

The effect of net charge on the solubility, activity, and stability of ribonuclease Sa.

The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.

Effect of beta-lactoglobulin and precipitation of calcium phosphate on the thermal coagulation of milk.

The effect of beta-lactoglobulin and heat-induced precipitation of calcium phosphate on the pH dependence and mechanism of thermal coagulation of milk throughout the pH range 6.3-7.3 was studied using serum protein-free milk and sodium caseinate as models for micellar and non-micellar milk protein systems respectively. It appears that the specific effect of beta-lactoglobulin at the pH of maximum stability may be related to its ability to chelate calcium. The effect of beta-lactoglobulin at the pH of minimum stability does not appear to be directly related to heat-induced dissociation of K-casein or micellar integrity but may be due to its ability to sensitize casein micelles to heat-induced precipitation of calcium phosphate, by increasing micellar hydrophobicity. The extent of heat-induced precipitation of calcium phosphate, as a function of pH, is an inverse reflection of the pH dependence of heat stability. Micellar integrity appears to play a critical role in the heat stability of milk but for reasons not previously appreciated.

Water-in-Carbon Dioxide Emulsions with Poly(dimethylsiloxane)-Based Block Copolymer Ionomers

The stability of water-in-CO2 (W/C) emulsions stabilized with poly(dimethylsiloxane)-b-poly(methacrylic acid) (PDMS-b-PMA) and PDMS-b-poly(acrylic acid) (PDMS-b-PAA) ionomer surfactants is reported as a function of surfactant architecture, pH, temperature, pressure, and droplet flocculation. For a given PDMS block length, the stability of the emulsion is correlated with the distance from the balanced state where the surfactant prefers the water and CO2 phases equally. When the pH starting at 3 is raised up to 5−6, the hydrophilic/CO2-philic balance of the surfactant increases, because of ionization of COOH, and the emulsion becomes more stable. At the pH of maximum stability, the emulsion becomes more stable with a decrease in the PDMS length, for a given ratio of block lengths, because of gelation of the flocculated 2−5 μm primary droplets. W/C emulsions are stable with respect to sedimentation for >24 h and are resistant to coalescence for more than 7 days. Because of gelation, the W/C emulsions are more stable than water/hexane emulsions (at ambient pressure) formed at the same conditions. The addition of 20% hexane to CO2 as a cosolvent reduced flocculation in some cases to zero.

Isomerization and Formulation Stability of the Vaccine Adjuvant QS-21

The stability of the immunologic adjuvant QS-21 (Cambridge Biotech Corp.) was optimized for use in the MN rgp120 HIV-1 subunit vaccine. QS-21, a saponin purified by reversed phase HPLC from an extract of the bark of the Quillaja saponaria Molina tree, consisted initially of one species (QS-21A), but converted to two species, QS-21A and QS-21 B, in aqueous solution. NMR studies indicated that the two species are structural isomers and that isomerization occurs by intramolecular trans-esterification of the fatty acid moiety between the 3- and 4-hydroxyl groups of the fucose ring (Jacobsen et al. Carbohydr. Res., in press). Both isomers were adjuvant active. Storage of QS-21 in aqueous solution resulted in the interconversion between these isomer forms, as well as the slow formation of degradation products due to ester hydrolysis. The critical micellar concentration of QS-21 in succinate buffer was measured by a fluorescent probe method to be 51 ± 9 μg/mL Studies were performed at different concentrations of QS-21 to assess the influence of micelle formation on stability. These experiments indicated that QS-21 is more stable in the micellar form, presumably because the most labile ester bond linking the fatty acid moiety to fucose is constrained or buried in the hydrophobic micellar environment. The pH of maximum stability was pH 5.5, the pH for minimum degradation of most esters. The final formulation, 500 μg/mL QS-21 in 20 mM sodium succinate, 150 mM NaCI, pH 5.5, provided a shelf-life of greater than 2 years.

Degradation kinetics of DMP 777, an elastase inhibitor.

The objective was to evaluate the degradation profile of the elastase inhibitor DMP 777 and lay the foundation for formulation development. The pKa was determined by potentiometric titration in mixed-aqueous solvents. The degradation kinetics were studied as a function of pH, buffer concentration, ionic strength, methanol concentration and temperature using a stability-indicating HPLC assay. The degradation products were identified by LC-MS, NMR, and by comparison with authentic samples. The pKa for the protonated piperazine nitrogen was estimated to be 7.04. The pH-rate profile is described by specific acid-, water-, and specific base-catalyzed pathways. The pH of maximum stability is in the range of 4 to 4.5 where water is the principal catalyst in the reaction. Buffer catalysis, primary salt effects and medium effects were observed. The proposed mechanism for acid catalyzed degradation is the rarely observed AAL1 which involves alkyl-nitrogen heterolysis. The driving force for the reaction appears to lie in the stability of the benzylic carbocation. The proposed mechanism for base catalyzed degradation is BAC2 which involves beta-lactam ring opening. The beta-lactam ring of DMP 777, a monolactam, appears to be as reactive as that in benzylpenicillin in the KOH controlled region where a similar mechanism of hydrolysis should be operative. A contributing factor to this increased reactivity may lie in the reduced basicity of the beta-lactam nitrogen making it a good leaving group. The degradation profile indicates that development of a solution dosage form of DMP 777 with adequate shelf-life stability at room temperature is feasible.

Glycolamide esters of 6-methoxy-2-naphthylacetic acid as potential prodrugs - physicochemical properties, chemical stability and enzymatic hydrolysis

Various glycolamide ester prodrugs of 6-MNA (I), an active metabolite of nabumetone (II), were synthesised to obtain NSAIDs with improved therapeutic index. To assess their suitability as prodrugs, these derivatives were evaluated for physicochemical properties, chemical stability and enzymatic hydrolysis in 80% human plasma. A number of these ester derivatives possessed physicochemical properties required for oral absorption (log P ≥ 2, solubility ≥ 10 μg/ ml). These esters were sufficiently stable in 0.05 M phosphate buffer of pH 7.4 at 37°C (half-lives 16–473 h). The disubstituted glycolamide esters XII-XXII were more stable than monosubstituted glycolamide esters IV-XI. Compound XII was also subjected to hydrolytic studies over the pH range 1.2–9.0 at 70°C and from the pH-rate profile, the pH of maximum stability was found to be 4.34 (4–5). At pH 4.5 compound XII showed a shelf life of 1.12 years at 20°C as determined from accelerated studies using the Arrhenius equation. The ester derivatives were quantitatively converted to 6-MNA very rapidly in human plasma at 37°C and pH 7.4 Comparison with chemical stability data shows that the intrinsic reactivity of esters has no effect on their enzymatic reactivity. The chemically most stable N,N-disubstituted glycolamide esters were better substrates for plasma enzymes (half-lives 7–83 s) compared to the monosubstituted glycolamide esters (half-lives 168–809 s). It is concluded that these derivatives possess many of the ideal properties of prodrugs and esterification of 6-MNA with substituted 2-hydroxyacetamides may be a potentially useful prodrug approach.

The influence of multivalent salts on the processability of a stearic acid monolayer: a stability, electron spectroscopy for chemical analysis and atomic force microscopy study

This work is focused on the enhancement of the monolayer stability by studying the pH-dependent adsorption of metal ions of varying valence to a stearic acid monolayer. The stability of the monolayer was detected by measuring the time-dependent change in the mean molecular area at a constant surface pressure. A rather narrow ion-specific pH range of maximum stability was found. Within this pH range, the monolayer acted almost as an ideal film and the molecular area at a predetermined surface pressure remained constant for several hours. The stability was correlated with the degree of dissociation of the monolayers which was determined by X-ray photoelectron spectroscopy. The observed p K a values were 4.3 for Tb stearate, 5.6 for Cd stearate and 6.2 for Mn stearate. The deposited multilayer structures were analyzed by atomic force microscopy. The films deposited at a pH of poor stability clearly contained more defects than the films fabricated at a pH of maximum stability. The resolution of the images was sufficient for a detailed crystallographic analysis of the molecular packing.

The pH-stability and acid degradation of the macrolide antibiotic, josamycin

The influence of pH, ionic strength and buffer concentration on the stability of josamycin has been investigated. The pH-rate profile for josamycin over the pH range 1–12 was characterised and the equation describing the profile determined. Results indicated that josamycin is subject to specific acid catalysis whilst catalysis in alkali media appeared to be more complex. The rate constant for catalysis by hydronium ion ( k H) was 54.11 M −1 · h −1 and the rate constant for catalysis by hydroxide ion ( k OH) was 60.35 M −1 · h −1. Catalysis due to water was insignificant and the water catalysed rate constant was found to be 3.37 × 10 −5 h −1. The pH of maximum stability was determined as pH 6.5 whilst degradation at pH 1.0 and 12.0 is about five orders of magnitude greater than at pH 6.5. The degradation of josamycin in acid is subject to a significant primary salt effect; however, no secondary salt effect was evident. Concentration vs. time profiles for josamycin in acidic media were biphasic which indicated that the degradation reaction did not follow a simple pathway whereby josamycin degrades directly to products. Further investigations suggest that josamycin undergoes a reversible isomerisation step, with subsequent degradation of josamycin and possibly its isomer, by cleavage of the mycarose moiety to desmycarose compounds. Studies to determine the stability of josamycin in simulated gastric fluids demonstrated that acid degradation could be appreciable after oral administration. However, extensive degradation in vivo will only occur at the most acidic gastric pH's of about pH 1.0 to 2.0.

Development of a stable oral liquid dosage form of spironolactone.

A clear, stable, oral liquid dosage form of spironolactone has been developed. Solubility profiles of spironolactone were obtained in several co-solvent blends. Using this data, a co-solvent blend containing polyethylene glycol 400 (30% v/v), propylene glycol (10% v/v), glycerin (10% v/v) and ethyl alcohol (10% v/v) was used to solubilize spironolactone at a concentration of 2 mg/ml. The final formulation contained sweetening agents (sucrose, saccharin sodium), flavours (cherry, sweet), a desensitizing agent (menthol), a dye (FD&C Red #40) and a preservative (benzoic acid) to incorporate the desired organoleptic and preservative properties. A phosphate buffer was used to maintain a pH value of 4.5 (pH of maximum stability as reported earlier) to minimize hydrolysis. The final dosage form was stable for at least 93 days at 40 degrees C (loss of potency less than 4%). According to FDA guidelines, a tentative expiration date of 2 years at 25 degrees C is justifiable.

Aqueous stability and solubility of CI-988, a novel "dipeptoid" cholecystokinin-B receptor antagonist.

The aqueous solubility and solution stability of the N-methylglucamine and sodium salts of CI-988 (CI-988 NMG and CI-988 Na) were evaluated to aid in the development of a parenteral formulation for preclinical and clinical testing. CI-988 ([R-(R*,R*)]-4-[[2-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2- [[(tricyclo[3.3.1.1(3,7)]dec-2-yloxy)- carbonyl]amino]propyl]amino]-1-phenylethyl]amino]-4-oxobutanoic acid) is a selective "dipeptoid" cholecystokinin-B receptor antagonist. The shape of the pH-solubility profile, generated at 30 degrees C, is consistent with the ionization of the terminal carboxyl group (pKa of 4.34). The pH-rate profile is independent of the salt form and is well described by two reaction pathways: spontaneous or water-catalyzed degradation of the nonionized form and specific base-catalyzed catalyzed degradation of the ionized form. The primary mechanism of degradation from the former pathway is consistent with intramolecular, carboxyl-assisted, amide-bond cleavage, whereas the primary mechanism of degradation from the latter pathway appears to be intramolecular cyclization to a hydantoin product with expulsion of 2-adamantanol. The pH dependencies of the solubility and stability show that a simple aqueous buffered solution of CI-988 has a predicted t90 of 2.1 years and a solubility of 0.94 mg/ml at pH 6.5, the theoretical pH of maximum stability, and 30 degrees C.

Stability of carmethizole hydrochloride (NSC-602668), an experimental cytotoxic agent

The solution stability and degradation mechanism of Carmethizole HCl ( 1, NSC-602668), an experimental anti-cancer drug, were studied. An apparent p K a value of 4.2 at 25°C and ionic strength = 0.15 M (NaCl) was determined for 1 by potentiometric titration. The degradation 1 in aqueous solution proceeds by two consecutive hydrolysis reactions of the bis-carbamate moieties at C4′ and C5′, with hydrolysis at C5′ hydrolyzing approximately 8 times faster than hydrolysis at C4′. The rates of degradation of 1 in aqueous solution were determined as a function of pH, temperature, buffer concentration, ionic strength, and co-solvent concentration. The pH of maximum stability was at pH < 2 for the degradation of 1 at 25°C. Analysis of the hydrolysis products by electron impact mass spectrometry showed that one 18O atom was incorporated into the first degradation product, 2, when the reaction was run in 18O-enriched water. This is consistent with alkyl C-O bond cleavage rather than an addition-elimination reaction at a carbamate group. The entropy of activation ( ΔS ‡ ) was −6.71 ± 0.05 e.u.at pH 9.90, indicating a unimolecular mechanism. All observations were consistent with an SN1 mechanism.

Chemical and enzymatic degradation of ganciclovir prodrugs: enhanced stability of the diadamantoate prodrug under acid conditions.

We report the chemical and enzymatic hydrolysis of two hydrophobic prodrugs of ganciclovir (3 = dipropionate ester; 4 = diadamantoate ester). Both prodrugs undergo hydrolysis showing a pH dependence of kobs = kH+aH+ + ko + kHO-aHO- and a pH of maximum stability near pH 5. Only 4 exhibited a shelf life (t90) greater than 2 years. Compound 4 reacts significantly slower than ganciclovir in acidic media, even though the adamantyl esters provide additional reaction sites (which would be expected to increase the rate of degradation) that are distally removed from the guanine ring system, offering negligible steric or electronic substituent effects. Both 3 and 4 hydrolyzed in tissue homogenate, where kobs followed liver greater than intestine much greater than skin. Based on these findings of chemical stability, lipophilicity, and acceptable rate of enzymatic cleavage by skin esterases, 4 meets several of the criteria required for the topical sustained delivery of ganciclovir.

Development of Oral Liquid Dosage Forms of Acetazolamide

Two oral liquid dosage forms of acetazolamide have been developed. Using the solubility profiles, polyethylene glycol 400 (7%, v/v) was used as the solubilizing agent and propylene glycol (53%, v/v) as the cosolvent to keep acetazolamide in solution. Because of the bitter taste of acetazolamide, sweetening agents (simple syrup, sorbitol solution, and artificial sweeteners) and flavors (raspberry, sweet, and menthol) were added to the final formulations. A buffer (either phosphate or citrate) solution was used to maintain a pH value of 4 (pH of maximum stability as reported earlier) to minimize hydrolysis. The final dosage forms were stable for at least 90 days at 37 °C (loss of potency of 5%). According to FDA guidelines, a tentative expiry date of 2 years at 25 °C is justifiable.

High-performance liquid chromatographic (HPLC) and HPLC-mass spectrometric (MS) analysis of the degradation of the luteinizing hormone-releasing hormone (LH-RH) antagonist RS-26306 in aqueous solution.

The kinetics of the degradation of an LH-RH antagonist, RS-26306,1, in aqueous solution from pH 1 to pH 11 were studied by reverse-phase HPLC. The pH-rate profiles at 50, 60, and 80 degrees C were U-shaped with the rate law of kobs = kHaH + kw + kOHaOH. The predicted 25 degrees C shelf life at the pH of maximum stability, pH approximately 5, is greater than 10 years. The products from the degradation were analyzed by HPLC-MS using thermospray ionization. Below pH 3, the primary product, 2, forms from the acid-catalyzed deamidation of the C-terminal amide. Above pH 7, epimerization of the individual amino acids is the principal reaction. Between pH 4 and pH 6, intramolecular serine-catalyzed peptide hydrolysis becomes important, yielding a tripeptide, 3, and a heptapeptide, 4. At the pH of maximum stability all three pathways for degradation are observed.

Conformational stability and mechanism of folding of ribonuclease T1

Urea and thermal unfolding curves for ribonuclease T1 (RNase T1) were determined by measuring several different physical properties. In all cases, steep, single-step unfolding curves were observed. When these results were analyzed by assuming a two-state folding mechanism, the plots of fraction unfolded protein versus denaturant were coincident. The dependence of the free energy of unfolding, delta G (in kcal/mol), on urea concentration is given by delta G = 5.6 - 1.21 (urea). The parameters characterizing the thermodynamics of unfolding are: midpoint of the thermal unfolding curve, Tm = 48.1 degrees C, enthalpy change at Tm, delta Hm = 97 kcal/mol, and heat capacity change, delta Cp = 1650 cal/mol deg. A single kinetic phase was observed for both the folding and unfolding of RNase T1 in the transition and post-transition regions. However, two slow kinetic phases were observed during folding in the pre-transition region. These two slow phases account for about 90% of the observed amplitude, indicating that a faster kinetic phase is also present. The slow phases probably result from cis-trans isomerization at the 2 proline residues that have a cis configuration in folded RNase T1. These results suggest that RNase T1 folds by a highly cooperative mechanism with no structural intermediates once the proline residues have assumed their correct isomeric configuration. At 25 degrees C, the folded conformation is more stable than the unfolded conformations by 5.6 kcal/mol at pH 7 and by 8.9 kcal/mol at pH 5, which is the pH of maximum stability. At pH 7, the thermodynamic data indicate that the maximum conformational stability of 8.3 kcal/mol will occur at -6 degrees C.