Effects of Insulin Concentration and Self-Association on the Partitioning of Its A-21 Cyclic Anhydride Intermediate to Desamido Insulin and Covalent Dimer

Purpose. In the pH range 2–5, human insulin degrades via deamidation at the A-21 asn and covalent dimerization. Both products form via a common cyclic anhydride intermediate, a product of intramolecular nucleophilic attack by the A-21 carboxyl terminus. This study examines the influence of [insulin] and self-association on the partitioning of the intermediate to products. Methods. Insulin self-association was characterized (pH 2–4) by concentration difference spectroscopy. Deamidation rates (pH 2–4) and concurrent rates of covalent dimer formation (pH 4) were determined versus [insulin] at 35°C by initial rates. A mathematical model was developed to account for the overall rate and product composition profile versus pH and [insulin]. Results. Between pH 2–4, insulin self-associates to form non-covalent dimers with a pH independent association constant of 1.8 × 10⁴ M ^–1. The overall rate of degradation is governed by intermediate formation, while product distribution is determined by competition between water and the phe B-l amino group of insulin for the anhydride. In dilute solutions, deamidation is first-order in [insulin] while covalent dimerization is second-order. Thus, deamidation predominates in dilute solutions but the fraction of covalent dimer formed increases with [insulin]. At high [insulin], self-association inhibits covalent dimer formation, preventing exclusive degradation via this pathway. The model accurately predicts a maximum in covalent dimer formation near pH 4. Conclusions. A mechanism is described which accounts for the complex dependence of insulin's degradation rate and product distribution profile on pH (between 2–5) and [insulin]. If these results can be generalized, they suggest that covalent aggregation in proteins may be inhibited by self-association.     

The Stability of Insulin in Crystalline and Amorphous Solids: Observation of Greater Stability for the Amorphous Form

Purpose. Generalizations based upon behavior of small molecules have established that a crystalline solid is generally much more stable toward chemical degradation than is the amorphous solid. This study examines the validity of this generalization for proteins using biosynthetic human insulin as the model protein. Methods. Amorphous insulin was prepared by freeze drying the supernate from a suspension of zinc insulin crystals adjusted to pH 7.1. Storage stability at 25°C and 40°C were compared for the freeze dried material, the dried suspended crystals, and the starting batch of crystals. Samples were equilibrated at selected relative humidities between zero and 75% to obtain samples at various water contents. Assays for dimer formation were performed by size exclusion HPLC and assays for deamidated product were carried out by reverse phase HPLC. Degradation was found to be linear in square root of time, and the slopes from % degradation vs. square root of time were used to define the rate constants for degradation. Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) were used to characterize the state of the protein in the solids. Results. As expected based upon previous results, the primary degradation pathways involve deamidation at the Asn^A21 site and co-valent dimer formation, presumably involving the A-21 site. Contrary to expectations, amorphous insulin is far more stable than crystalline insulin under all conditions investigated. While increasing water content increases the rate of degradation of crystalline insulin, rate constants for degradation in the amorphous solid are essentially independent of water content up to the maximum water content studied (15%). Conclusions. Based upon the FTIR and DSC data, both crystalline and amorphous insulin retain some higher order structure when dried, but the secondary structure is significantly perturbed from that characteristic of the native solution state. However, neither DSC nor FTIR data provide a clear interpretation of the difference in stability between the amorphous and crystalline solids. The mechanism responsible for the superior stability of amorphous insulin remains obscure.     

Chemical Stability of Insulin. 1. Hydrolytic Degradation During Storage of Pharmaceutical Preparations

Hydrolysis of insulin has been studied during storage of various preparations at different temperatures. Insulin deteriorates rapidly in acid solutions due to extensive deamidation at residue Asn^A21. In neutral formulations deamidation takes place at residue Asn^B3 at a substantially reduced rate under formation of a mixture of isoAsp and Asp derivatives. The rate of hydrolysis at B3 is independent of the strength of the preparation, and in most cases the species of insulin, but varies with storage temperature and formulation. Total transformation at B3 is considerably reduced when insulin is in the crystalline as compared to the amorphous or soluble state, indicating that formation of the rate-limiting cyclic imide decreases when the flexibility of the tertiary structure is reduced. Neutral solutions containing phenol showed reduced deamidation probably because of a stabilizing effect of phenol on the tertiary structure (-helix formation) around the deamidating residue, resulting in a reduced probability for formation of the intermediate imide. The ratio of isoAsp/Asp derivative was independent of time and temperature, suggesting a pathway involving only intermediate imide formation, without any direct side-chain hydrolysis. However, increasing formation of Asp relative to isoAsp derivative was observed with decreasing flexibility of the insulin three-dimensional structure in the formulation. In certain crystalline suspensions a cleavage of the peptide bond A8–A9 was observed. Formation of this split product is species dependent: bovine > porcine > human insulin. The hydrolytic cleavage of the peptide backbone takes place only in preparations containing rhombohedral crystals in addition to free zinc ions.To whom correspondence should be addressed at Nove Research Institute, Novo Alle, DK-2880Bagsvaerd, Denmark     

Insulin Aggregation in Aqueous Media and Its Effect on Alpha-Chymotrypsin-Mediated Proteolytic Degradation

Self-association of zinc–insulin monomers into dimers and hexamers may lead to enhanced protection of the peptide from proteolytic degradation. The present study has been undertaken to investigate the relationship, if any, between the rate of enzymatic degradation of insulin by a protease, alpha-chymotrypsin, and the extent of insulin aggregation in aqueous solutions. Insulin solutions (0.6 mg/ml) containing varying proportions of dimer and hexamer were obtained by adding ethylene diamine tetraacetic acid (EDTA) within a concentration range of 0.005 to 0.040 mM. As the EDTA concentration was increased above 0.040 mM, a complete dissociation of hexamers to dimers occurred and the rate of enzymatic degradation reached its maximum. The overall first-order rate constants appeared to be linearly related to the square of EDTA concentrations. The apparent first-order rate constants for dimer and hexamer degradation obtained from a linear plot of rate constant versus EDTA squared concentration were found to be 0.02800 ± 0.00065 and 0.00798 ± 0.00075 min^–1, respectively. Two major insulin degradation products were also detected and the kinetics of product appearance agreed well with the disappearance kinetics of insulin. The results indicated that the degradation of insulin dimers by alpha-chymotrypsin is about 3.5 times faster than the degradation of the hexamer. The second-order dependency of degradation rate on EDTA concentration might be due to the fact that insulin hexamers contain two zinc ions which are sequestered by two EDTA molecules. Chelation of zinc ions by EDTA lead to hexamer deaggregation to dimers as was evidenced from a circular dichroism study. Formation of three dimer species from one hexamer aggregate should theoretically enhance the rate of degradation threefold, a value consistent with the experimentally determined ratio of 3.5.     

Isolation and Identification of Peptide Degradation Products of Heat Stressed Pramlintide Injection Drug Product

Purpose. This report summarizes the identification of nine deamidation and four hydrolysis products from a sample of pramlintide injection final drug product that was subjected to stress at 40°C for 45 days. Methods. The pramlintide degradation products were isolated by strong cation exchange HPLC followed by reversed-phase HPLC. Subsequent to isolation, the molecular weight of each component was determined by liquid chromatography-mass spectrometry (LC/MS). Further characterization was accomplished by amino acid sequence analysis and/ or enzymatic (thermolysin) digestion followed by LC/MS and sequence analysis. Results. The isolated products were identified as [iso-Asp²¹]-pramlintide, [iso-Asp³]-pramlintide, and [iso-Asp²²]-pramlintide, the deamidation products of pramlintide with rearrangement at Asn²¹, Asn³, and Asn²², respectively. Also found were [Asp/iso-Asp¹⁴]-pramlintide, and [Asp/iso-Asp³⁵]-pramlintide, the deamidation products at Asn¹⁴, and Asn³⁵, and [Asp²¹]-pramlintide together with [Asp²²]-pramlintide. For the deamidations at the 14^th and 35^th residues, it could not be determined whether the substance corresponded to the Asp or the iso-Asp product. The [Asp²¹] and [Asp²²] products could not be separated from each other chromatographically but were both identified in a single fraction. Two minor degradation products were also identified as deamidated species. However, the sites of deamidation remain unknown. Also identified were [l-18]-pramlintide, [l-19]-pramlintide, [19-37]-pramlintide, and [20-37]-pramlintide, the products of hydrolytic peptide backbone cleavage at amino acids His¹⁸/Ser¹⁹ and Ser¹⁹/Ser²⁰, respectively. One other product was isolated and tentatively identified as a cyclic imide intermediate preceeding deamidation. Conclusions. The primary mode of thermally induced degradation for this peptide is deamidation. A second degradation mechanism is peptide backbone hydrolysis.     

Effect of Ethylenediamine on Chemical Degradation of Insulin Aspart in Pharmaceutical Solutions

Purpose  To examine the effect of different amine compounds on the chemical degradation of insulin aspart at pharmaceutical formulation conditions. Methods  Insulin aspart preparations containing amine compounds or phosphate (reference) were prepared and the chemical degradation was assessed following storage at 37°C using chromatographic techniques. Ethylenediamine was examined at multiple concentrations and the resulting insulin–ethylenediamine derivates were structurally characterized using matrix assisted laser desorption ionization time-of-flight mass spectroscopy. The effects on ethylenediamine when omitting glycerol or phenolic compounds from the formulations were investigated. Results  Ethylenediamine was superior in terms of reducing formation of high molecular weight protein and insulin aspart related impurities compared to the other amine compounds and phosphate. Monotransamidation of insulin aspart in the presence of ethylenediamine was observed at all of the six possible Asn/Gln residues with Asn^A21 having the highest propensity to react with ethylenediamine. Data from formulations studies suggests a dual mechanism of ethylenediamine and a mandatory presence of phenolic compounds to obtain the effect. Conclusions  The formation of high molecular weight protein and insulin aspart related impurities was reduced by ethylenediamine in a concentration dependant manner.     

Assembly and Dissociation of Human Insulin and LysB28ProB29-Insulin Hexamers: A Comparison Study

Purpose. Investigations into the kinetic assembly and dissociation of hexameric Lys^B28Pro^B29-human insulin (LysPro), a rapid-acting insulin analog produced by the sequence inversion of amino acids at positions B28 and B29, were designed to explain the impact that the sequence inversion has on the formulation and pharmacokinetics of the insulin analog. Methods. The kinetics of phenolic ligand binding to human insulin and LysPro were studied by stopped-flow spectroscopy. The kinetics of R₆ hexamer disruption were studied by extraction of Co(II) with EDTA. Results. Phenolic ligand binding to human insulin yielded rate constants for a fast and slow phase that increased with increasing ligand concentration and are attributed to the T₆ T₃R₃ and T₃R₃ R₆ transitions, respectively. However, the kinetics of phenolic ligand binding with LysPro was dominated by rates of hexamer assembly. The kinetic differences between the insulin species are attributed to alterations at the monomer-monomer interface in the dimer subunit of the LysPro analog. The extraction of Co(II) from both hexameric complexes by EDTA chelation is slow at pH 8.0 and highly dependent on ligand concentration. Cobalt extraction from LysPro was pH dependent. Of the various phenolic ligands tested, the relative affinities for binding to the human and LysPro hexamer are resorcinol > phenol > m-cresol. Conclusions. The extraction data support the formation of an R₆-type LysPro hexamer under formulation conditions, i.e., in the presence of divalent metal and phenolic ligand, that is similar in nature to that observed in insulin. However, the formation kinetics of LysPro identify a radically different monomeric assembly process that may help explain the more rapid pharmacokinetics observed with the hexameric formulation of LysPro insulin relative to human insulin.     

Aqueous Stability of Human Epidermal Growth Factor 1-48

Human epidermal growth factor 1-48 (hEGF 1-48, Des(49-53)hEGF) is a single chain polypeptide (48 amino acids; 3 disulfide bonds; 5445 Da) possessing a broad spectrum of biologic activity including the stimulation of cell proliferation and tissue growth. In this study, three primary aqueous degradation products of hEGF 1-48 were isolated using isocratic, reverse phase/ion-pair HPLC. The degradation products were characterized using amino acid sequencing, electrospray ionization mass spectrometry, isoelectric focusing, and degradation kinetics. Results indicate that hEGF 1-48 degrades via oxidation (Met²¹), deamidation (Asn¹), and succinimide formation (Asp¹¹). The relative contribution of each degradation pathway to the overall stability of hEGF 1-48 changes as a function of solution pH and storage condition. Succinimide formation at Asp¹¹ is favored at pH < 6 in which aspartic acid is present mostly in its protonated form. Deamidation of Asn¹ is favored at pH > 6. The relative contribution of Met²¹ oxidation is increased with decreasing temperature, storage as a frozen solution (–20°C), and exposure to fluorescent light.     

Stabilization of pH-Induced Degradation of Porcine Insulin in Biodegradable Polyester Microspheres

The purpose of this research project was to stabilize the pH-induced degradation of porcine insulin encapsulated within biodegradable polyester microspheres through the incorporation of a basic additive. Insulin microspheres fabricated using Poly(L-lactide) (L-PLA) and Poly(DL-lactide-co-glycolide) (50:50 DL-PLGA) were subjected to in vitro release studies and the stability of unreleased insulin encapsulated within microspheres was investigated. The intramicrosphere pH was estimated by encapsulating acid-base indicators covering a wide pH transition range within 50:50 DL-PLGA microspheres. Finally, a basic excipient sodium bicarbonate was incorporated in 50:50 DL-PLGA microspheres to minimize acid-induced insulin degradation. The in vitro release was slow and incomplete (<30% in 30 days). Extraction and analyses of the unreleased insulin within the microspheres revealed that an average of ～11% remained intact. The degradation products observed consisted of ～15% of three distinct deamidated hydrolysis products including A-21 Desamido insulin, ～22% Covalent Insulin Dimer and trace amounts of High Molecular Weight Transformation Products. Comparison of the degradation profile of unreleased insulin contained in various microsphere formulations with the in vitro release kinetics indicated that an increase in covalent dimer formation within the microspheres prior to release is associated with a decrease in the cumulative percent insulin released during a 30-day incubation period. In an attempt to correlate insulin degradation with the drop in intra-microsphere pH due to polymer hydrolysis, it was determined that the pH within a degrading microsphere reaches a value of ～1.8 after 4 weeks. The incorporation of a basic excipient, sodium bicarbonate, in 50:50 DL-PLGA microspheres resulted in an improved in vitro release profile (cumulative release ～47.3% in 30 days) as well as a significant reduction in covalent dimerization of the unreleased insulin to barely detectable levels. The low pH microenvironment within a degrading microsphere is one of the major factors leading to protein instability, and the degradation of proteins encapsulated within polyester microspheres can be minimized by the incorporation of a basic excipient.     

Towards Heat-stable Oxytocin Formulations: Analysis of Degradation Kinetics and Identification of Degradation Products

Purpose  To investigate degradation kinetics of oxytocin as a function of temperature and pH, and identify the degradation products. Materials and Methods  Accelerated degradation of oxytocin formulated at pH 2.0, 4.5, 7.0 and 9.0 was performed at 40, 55, 70 and 80°C. Degradation rate constants were determined from RP-HPLC data. Formulations were characterized by HP-SEC, UV absorption and fluorescence spectroscopy. Degradation products were identified by ESI-MS/MS. Results  The loss of intact oxytocin in RP-HPLC was pH- and temperature-dependent and followed (pseudo) first order kinetics. Degradation was fastest at pH 9.0, followed by pH 7.0, pH 2.0 and pH 4.5. The Arrhenius equation proved suitable to describe the kinetics, with the highest activation energy (116.3 kJ/mol) being found for pH 4.5 formulations. At pH 2.0 deamidation of Gln⁴, Asn⁵, and Gly⁹-NH₂, as well as combinations thereof were found. At pH 4.5, 7.0 and 9.0, the formation of tri- and tetrasulfide-containing oxytocin as well as different types of disulfide and dityrosine-linked dimers were found to occur. Beta-elimination and larger aggregates were also observed. At pH 9.0, mono-deamidation of Gln⁴, Asn⁵, and Gly⁹-NH₂ additionally occurred. Conclusions  Multiple degradation products of oxytocin have been identified unequivocally, including various deamidated species, intramolecular oligosulfides and covalent aggregates. The strongly pH dependent degradation can be described by the Arrhenius equation.     

Chemical Stability of Insulin. 2. Formation of Higher Molecular Weight Transformation Products During Storage of Pharmaceutical Preparations

Formation of covalent, higher molecular weight transformation (HMWT) products during storage of insulin preparations at 4–45°C was studied by size exclusion chromatography. The main products are covalent insulin dimers (CID), but in protamine-containing preparations the concurrent formation of covalent insulin-protamine (CIP) products takes place. At temperatures 25°C parallel or consecutive formation of covalent oligo- and polymers can also be observed. Rate of HMWT is only slightly influenced by species of insulin but varies with composition and formulation, and for isophane (NPH) preparations, also with the strength of preparation. Temperature has a pronounced effect on CID, CIP, and, especially, covalent oligo- and polymer formation. The CIDs are apparently formed between molecules within the hexameric unit common for all types of preparations and rate of formation is generally faster in glycerol-containing preparations. Compared with insulin hydrolysis reactions (see the preceding paper), HMWT is one order of magnitude slower, except for NPH preparations.To whom correspondence should be addressed at Nove Research Institute, Novo Alle, DK-2880Bagsvaerd, Denmark     

Adsorption of insulin with varying self-association profiles to a solid Teflon surface—Influence on protein structure, fibrillation tendency and thermal stability

Interfaces are present in the preparation of pharmaceutical products and are well known for having an influence on the physical stability of proteins. The aim of this study was to examine the conformation (i.e. secondary and tertiary structures) and fibrillation tendency, overall aggregation tendency and thermal stability of adsorbed human insulin at a solid particulate Teflon surface. The effects of changes in the association degree of insulin on the structure and stability have been determined. Using SEC-HPLC, association profiles were determined for insulin aspart, zinc-free human insulin and human insulin with two Zn²⁺ per hexamer in concentrations ranging from 0.1 mg/ml to 20 mg/ml. Insulin aspart was 100% monomeric, regardless of concentration. In contrast, human insulin went from 100% monomer to 80% hexamer, and 20% dimer/monomer and zinc-free human insulin from 100% monomer to 70% dimer and 30% monomer with increasing concentration. The secondary structure of the insulins changed upon adsorption, but only minor differences were observed among the insulins. Structural changes were observed when the insulin-surface ratio was varied, but at no point did the structure resemble that of fibrillated insulin in solution. The presence of particles resulted in increased fibrillation of human insulin. The lag-time of fibrillation decreased, when the amount of particles present was increased. In conclusion, the type and association degree of the three insulin variants has no major influence on the secondary structure observed after adsorption of insulin at the solid Teflon surface. However, the presence of particles increases the tendency of insulin to fibrillate.     

Effect of the three-dimensional structure on the deamidation reaction of ribonuclease A

Abstract: Kinetic data on the deamidation reaction of Asn⁶⁷ in RNase A and of Asn³ in the two peptides Ac-Cys-Lys-Asn-Gly-Gln-Thr-Asn-Cy s-NH₂ and Ac-Cys(Me)-Lys-Asn-Gly-Gln-Thr-Asn-Cys(Me)-NH₂, whose sequences are similar to that of the deamidation site in the enzyme, have been determined in a wide range of pH and buffer concentrations. The values of the observed rate constant (k) for the enzyme are markedly lower than those for the peptides. However, the k dependence on pH and buffers is similar for all three substrates, indicating a similar reaction mechanism. The lower k-values for the enzyme have been quantitatively related to the thermal stability and the three-dimensional structure of the enzyme.     

Porcine insulin biodegradable polyester microspheres: stability and in vitro release characteristics

The stability of porcine insulin in biodegradable polymers, i.e., poly(DL-lactide-co-glycolide) 50:50 (50:50 DL-PLGA) and poly(L-lactide) (L-PLA) was investigated. Insulin encapsulated microspheres were fabricated from both polymers using double-emulsion-solvent evaporation and emulsion-solvent evaporation techniques and subjected to accelerated stability studies at 40 degrees C and 75% relative humidity. Porcine insulin was found to degrade in all microsphere formulations with an average of < 50% of the initial loading amount remaining intact at the end of 4 weeks. The two major degradation products observed in these formulations were determined to be A-21 desamido insulin and covalent insulin dimer with trace amounts of high molecular weight transformation products. In vitro release studies in phosphate buffered saline at 37 degrees C resulted in very slow and incomplete (< 30% in 30 days) release kinetics for all microsphere formulations. Extraction and analyses of the unreleased insulin within the microspheres revealed that an average of approximately 11% of the encapsulated insulin remained intact. The degradation products observed consisted of approximately 15% of three distinct deamidated hydrolysis products including A-21 desamido insulin, approximately 22% covalent insulin dimer, and trace amounts of high molecular weight transformation products. The degradation of porcine insulin within biodegradable polyester microspheres during stability and release studies can be attributed to the gradual decrease in the pH within the microspheres due to progressive polymer hydrolysis resulting in the production of DL-lactic and glycolic acids. The encapsulation of an acid-base indicator, bromophenol blue, in 50:50 PLGA microspheres (as a probe to estimate pH within the microspheres during accelerated stability studies) indicated that the pH decreased to approximately 3.8 after 3 weeks.     

Insulin aspart (AspB28 human insulin) derivatives formed in pharmaceutical solutions

Purpose. To isolate and identify the main insulin aspart (Asp^B28 human insulin) derivatives formed in pharmaceuticals (pH 7.4 at 5°C), to estimate rates of formation, and to determine their biologic potencies. Methods. Insulin aspart derivatives have been isolated by reversed-phase high-performance liquid chromatography (RP-HPLC), and identified by RP-HPLC, peptide mapping, amino acid analysis, mass spectrometry, and N-terminal amino acid sequence analysis. Results. The main derivatives formed were isoAsp^B28, isoAsp^B3, Asp^B3, and desPhe^B1-N-oxalyl-Val^B2 insulin aspart. At 5°C, the rate constants were 0.00028/month for isoAsp^B28 and isoAsp^B3, 0.00024/month for Asp^B3, and 0.00013/month for desPhe^B1-N-oxalyl-Val^B2 derivatives of insulin aspart. Unexpectedly, the rate of isomerization of B28 was high compared to the rate of B3 deamidation at both 5°C and 45°C. The N-terminal and especially the C-terminal of the B-chain are highly flexible, which may explain the high rate of isoAsp^B28 formation and that deamidation of Asn^B3 occurs. All the derivatives had full in vivo biologic potencies. Conclusion. Except for isoAsp^B28 insulin aspart, the main derivatives formed in pharmaceuticals of insulin aspart and human insulin at pH 7.4 are similar. They are all fully active in vivo. In proteins, flexibility of the polypeptide chain seems more important than sequence in the formation of succinimides.     

Effects of Phenol and meta-Cresol Depletion on Insulin Analog Stability at Physiological Temperature

The stability of three commercial “fast-acting” insulin analogs, insulin lispro, insulin aspart, and insulin glulisine, was studied at various concentrations of phenolic preservatives (phenol and/or meta-cresol) during 9 days of incubation at 37°C. The analysis by both size-exclusion and reversed-phase chromatography showed degradation of lispro and aspart that was inversely dependent on the concentration of phenolic preservatives. Insulin glulisine was much more stable than the other analogs and showed minimal degradation even in the absence of phenolic preservatives. With sedimentation velocity ultracentrifugation, we determined the preservatives' effect on the insulins' self-assembly. When depleted of preservatives, insulin glulisine dissociates from higher molecular weight species into a number of intermediate molecular weight species, in between monomer and hexamer, whereas insulin aspart and insulin lispro dissociate into monomers and dimers. Decreased stability of insulin lispro and insulin aspart seems to be because of the extent of dissociation when depleted of preservative. Insulin glulisine's dissociation to intermediate molecular weight species appears to help minimize its degradation during incubation at 37°C. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2255–2267, 2014     

The Role of Intramolecular Nucleophilic Catalysis and the Effects of Self-Association on the Deamidation of Human Insulin at Low pH

The influence of intramolecular catalysis and self-association on the kinetics of deamidation at the A-21 Asn residue of human insulin was explored at low pH and 35°C. Observed rate constants of overall insulin degradation were determined as a function of pH over a pH range of 2.0–5.0 and as a function of total insulin concentration between pH 2.0–4.0. The pH-rate behavior of both monomeric and associated insulin degradation from pH 2.0 to 5.0 indicated intramolecular catalysis by the unionized carboxyl terminus of the A chain. Anhydride trapping with aniline at pH 3.0 provided evidence supporting the formation of a cyclic anhydride intermediate in the rate limiting step indicative of intramolecular nucleophilic catalysis. Insulin in the presence of aniline at low pH formed two anilide products, A-21 N²-phenyl asparagine and N²-phenyl aspartic acid human insulin, at the expense of desamido A-21 formation, consistent with the partitioning of a common intermediate. Self-associated insulin degraded at a rate approximately 2.5 times greater than that of the monomer at pH 2.0 and pH 3. However, self-association had a negligible or slight stabilizing effect on insulin decomposition at pH 4.0. An apparent downward shift in the pK_a of the carboxyl terminus of approximately 0.75 units upon self-association and a catalytic rate constant which increases with -COOH acidity are proposed to account for these observations.In partial fulfillment for a Ph.D. degree in the     

Kinetic and thermodynamic control of the relative yield of the deamidation of asparagine and isomerization of aspartic acid residues

Abstract: Selective deamidation of Asn⁶⁷ of RNase A to β‐Asp⁶⁷ and Asp⁶⁷ residues at neutral pH initially produces greater amounts of the β‐Asp derivative. As the reaction proceeds the relative concentration of [Asp⁶⁷]–RNase A increases and, at equilibrium, becomes predominant. Such a discrepancy between the kinetic and thermodynamic control on reaction products is discussed in light of information from X‐ray three‐dimensional analysis and the lower thermodynamic stability of the β‐Asp derivative relative to the parent enzyme.     

1H n.m.r. studies of insulin

¹H n.m.r. studies at 270 MHz were made of the transformation of 2 Zn insulin hexamer to 4 Zn hexamer produced by the addition of anions (thiocyanate ion). Four separate H2 histidine resonances were observed for the B5 and B10 histidines in 2 Zn hexamer at pH 7 and 9 and four separate resonances also occurred in the 4 Zn hexamer. The observation of these resonances and others from phenylalanine, tyrosine and leucine residues showed that the 2 Zn to 4 Zn transformation probably occurred in solution in a similar manner to that observed in the crystal. Furthermore as occurred in the crystal, it was found that in solution the transformation was reversible (on removal of thiocyanate) and that 2 Cd insulin was unable to undergo the transformation. Des-Phe-B1-insulin did not undergo the transformation. Addition of SCN? to Zn-free insulin (mainly dimer) produced only a small transformation, consistent with the idea that Zn^{2 +} promotes formation of hexamer from dimer but probably does not otherwise affect the transformation.     

Degradation of aspartic acid and asparagine residues in human growth hormone-releasing factor

Products of the degradation of human growth hormone-releasing factor (GRF) in aqueous solutions (15–200 μM) have been isolated and fully characterized. The cleavage product, GRF(4–44)-NH₂, and the isomer-ization product, [β-Asp³]GRF(1–44)-NH₂, from the degradation of GRF(1–44)-NH₂ in acidic solution and the corresponding products, GRF(4–29)-NH₂ and [β-Asp³]GRF(1–29)-NH₂, from the degradation of GRF(1–29)-NH₂ have been isolated and characterized. The products, [β-Asp⁸]GRF(1–44)-NH₂ and [Asp⁸]GRF(1–44)-NH₂, from the deamidation of GRF(1–44)-NH₂ at pH 8.0 and the corresponding products, [β-Asp⁸]GRF(1–29)-NH₂ and [Asp⁸]GRF(1–29)-NH₂, from the deamidation of GRF(1–29)-NH₂ have been isolated and characterized. AH the degradation products of GRF(1–44)-NH₂ and GRF(1–29)-NH₂ were evaluated for biological activity and found to have much lower in vitro potencies than the parent peptides. Degradation occurs at Asp³ and Asn⁸ and the kinetics of these various transformations versus pH and temperature have been studied. GRF is most stable at pH 4–5. At pH below the pK_a of the Asp³ side-chain (pH<4), cleavage at Asp³-Ala⁴ is the major route of degradation. For pH>4, isomerization of Asp³ to β-Asp³ (iso-Asp³) predominates. The rates of cleavage and isomerization are simple first order and vary with pH, independent of buffer concentration, such that the protonated (COOH) form of Asp³ undergoes cleavage while the ionized (COO^-) form isomerizes. The more rapid deamidation of Asn⁸ to generate β-Asp⁸ and Asp⁸ in about a 4:1 ratio, presumably via a cyclic imide intermediate, occurs at pH < 5 and is general base-catalyzed. Evidence was also obtained for direct hydrolysis of protonated Asn⁸ in GRF(1–29)-NH₂ at pH<2 to give exclusively [Asp⁸]GRF(1–29)-NH₂. The deamidation of Asn⁸ in GRF(1–29)-NH₂ at pH 8.0, 22–55°C, is relatively insensitive to temperature for T>37°C, possibly due to conformational constraints. Asp²⁵ and Asn³⁵ are sterically, conformationally, or otherwise hindered with respect to these changes as no degradation at these sites was observed under the conditions employed.