Residual Proteolytic Activity Research Articles

Nitrogen Availability and Utilisation of Oligopeptides by Yeast in Industrial Scotch Grain Whisky Fermentation

Scotch grain whisky is produced with a substantial proportion of unmalted grains, which can result in nitrogen deficiency for yeast in the fermentable grain mash. This study examined nitrogen source availability and utilisation by three commercial whisky strains (Saccharomyces cerevisiae) during Scotch grain whisky fermentation, focusing on oligopeptides. Peptide uptake kinetics in synthetic whisky mash with defined peptides showed that oligopeptides of up to nine amino acids were taken up by the strains, albeit with some variability between the strains. The study found that peptides with appropriate molecular weights could replace free amino acids without negatively affecting fermentation kinetics. Moreover, fermentation performance improved when additional nitrogen was provided via peptides rather than diammonium phosphate. Analysis of industrial grain mash indicated that despite low initial yeast assimilable nitrogen, residual proteolytic activity from malt increased nitrogen availability during fermentation. Approximately 30% of the nitrogen consumed by yeast during grain mash fermentation was derived from peptides. LC-HRMS peptide analysis revealed complex dynamics of peptide formation, degradation, and utilisation. This study highlights the importance of oligopeptides in ensuring optimal fermentation efficiency in Scotch grain mash and similar substrates.

The Impact of Low-Temperature Inactivation of Protease AprX from Pseudomonas on Its Proteolytic Capacity and Specificity: A Peptidomic Study

The destabilization of UHT milk during its shelf life can be promoted by the residual proteolytic activity attributed to the protease AprX from Pseudomonas. To better understand the hydrolysis patterns of AprX, and to evaluate the feasibility of using low-temperature inactivation (LTI) for AprX, the release of peptides through AprX activity on milk proteins was examined using an LC-MS/MS-based peptidomic analysis. Milk samples were either directly incubated to be hydrolyzed by AprX, or preheated under LTI conditions (60 °C for 15 min) and then incubated. Peptides and parent proteins (the proteins from which the peptides originated) were identified and quantified. The peptides were mapped and the cleavage frequency of amino acids in the P1/P1′ positions was analyzed, after which the influence of LTI and the potential bitterness of the formed peptides were determined. Our results showed that a total of 2488 peptides were identified from 48 parent proteins, with the most abundant peptides originating from κ-casein and β-casein. AprX may also non-specifically hydrolyze other proteins in milk. Except for decreasing the bitterness potential in skim UHT milk, LTI did not significantly reduce the AprX-induced hydrolysis of milk proteins. Therefore, the inactivation of AprX by LTI may not be feasible in UHT milk production.

Use of Calotropis procera cysteine peptidases (CpCPs) immobilized on glyoxyl-agarose for cheesemaking

Calotropis procera cysteine peptidases (CpCPs) have presented several potential biotechnological applications. Here, these enzymes were immobilized on glyoxyl-agarose (glyoxyl-CpCPs) with yields of 90–95 % and the recovered activities ranged from 10 % to 15 %, according to enzyme loadings (5, 10, 20, 40, and 50 mgBSAeq/g). Spectrophotometric assays and SDS-PAGE showed that the casein hydrolysis by glyoxyl-CpCPs was similar to soluble CpCPs. In addition, glyoxyl-CpCPs exhibited similar ratio of milk-clotting activity to proteolytic activity in comparison with soluble CpCPs and chymosin. Even after being stored for six months at 8 °C, the residual proteolytic activity of glyoxyl-CpCPs remained close to 100 %. Atomic force microscopy and dynamic light scattering techniques showed that the process of casein micelle aggregation after treatment with glyoxyl-CpCPs was very similar to its soluble form and chymosin. Glyoxyl-CpCPs performed well after five reaction cycles, producing cheeses with yield, moisture, protein, and fat similar to those produced with chymosin.

Investigating the Short Peptidome Profile of Italian Dry-Cured Ham at Different Processing Times by High-Resolution Mass Spectrometry and Chemometrics.

Short peptides have been spiking interest owing to their significant contribution to the taste and functional properties of dry-cured ham. In this study, a suspect screening approach based on high-resolution mass spectrometry was employed for the comprehensive characterization of the short endogenous peptidome in dry-cured ham samples at different processing stages (14, 22, and 34 months). After careful manual spectra interpretation, a chemometric approach based on principal component analysis was employed for highlighting the differences between the three sets of samples. A total of 236 short peptide sequences was tentatively identified, including 173 natural short peptides and 63 sequences containing non-proteinogenic amino acids, the highest number ever reported for endogenous sequences in dry-cured ham. Samples in the latest processing stages presented a generally higher abundance of dipeptides, indicating residual proteolytic activity. Moreover, the several annotated modified short peptides, mainly pyroglutamination and lactoyl conjugation, allowed hypothesizing several reactions occurring over time. For the first time, several lactoyl-dipeptides were tentatively identified in dry-cured ham samples with maximum concentration in the late processing stage samples. The presented results significantly contribute to the understanding of the reaction involving short peptides that affect the sensory and functional properties of dry-cured ham.

Impact of thermal inactivation conditions on the residual proteolytic activity and the viscosity properties of whey protein concentrate enzymatic hydrolysates

The rationale for enzymatically hydrolysis of whey proteins includes enhancement of their technofunctional properties and the release of bioactive peptide sequences. This study investigated the impact of thermal treatments, i.e., heating using a water bath, using a tubular heat exchanger and heating during spray drying, on the residual proteolytic activity (RA), apparent viscosity (ηapp) and gelation temperature (Tg) of whey protein concentrate hydrolysates (WPH) generated with Alcalase® (WPH-Alc), Neutrase™ (WPH-Neu) and Debitrase® (WPH-Deb). The WPHs generated showed different degrees of hydrolysis (DH, 2.60–11.13%) and chromatographic profiles. Thermal inactivation studies showed different impacts on RA dependent on the thermal treatment conditions. The ηapp of unheated WPHs were significantly lower (16–18%) than that of the unhydrolysed whey protein concentrate (WPC). Thermal inactivation (80 °C waterbath) increased ηapp of all samples producing aggregates. Tubular heat exchanger inactivation increased ηapp of WPH-Alc and WPH-Deb at different rates, whereas no significantly changes were observed for WPH-Neu for holding times lower than 10 min. At 45% (w/v) total solids (TS) the viscosity of WPC was 1.0 Pa s, whereas the equivalent viscosity for WPC -Alc and WPH-Deb was achieved at 49.5 and 50.1% TS. Different trends were observed in Tg of the WPHs which were dependent on the enzyme and thermal treatment. Overall, this study demonstrates that judicious choice of thermal treatments during WPH enzyme inactivation is required in order to achieve an appropriate balance between RA, ηapp and Tg.

Investigation of the effect of b-galactosidase enzyme on the storage capacity of low-lactose milk

The article discusses the effect of the b-galactosidase enzyme on the storage capacity of milk with hydrolyzed lactose. For this purpose, parallel studies of a sample of sterilized milk and low-lactose milk, produced on its basis, were carried out. The peptide profile was used as a criterion for assessing the storage capacity of milk that underwent enzymatic decomposition of lactose. Assessment of the state of the peptide profile during storage was recorded at three control points — 30, 60 and 90 days. Studies have shown that at the second checkpoint, peptides were identified that characterize the proteolytic activity in the product. The third checkpoint study revealed the presence of low molecular weight peptides responsible for the bitter taste in milk. Parallel studies of sterilized milk without the addition of the enzyme did not reveal any changes in the peptide profile. The studies carried out indicate that the b-galactosidase enzyme has a residual proteolytic activity, which negatively affects the storage capacity of low-lactose milk and, as a consequence, the products, produced on its basis.

Sudden anaphylactic death: new insights to identify allergens involved

A woman in her early 20s died suddenly after dinner at a restaurant. She had been served a meal consisting of cream of pea, bruschetta and pasta with wild boar...

Modifying ADAMTS13 to modulate binding of pathogenic autoantibodies of patients with acquired thrombotic thrombocytopenic purpura.

Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura commonly target the spacer epitope R568/F592/R660/Y661/Y665 (RFRYY) of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). In this study we present a detailed contribution of each residue in this epitope for autoantibody binding. Different panels of mutations were introduced to create a large collection of full-length ADAMTS13 variants comprising conservative (Y← →F), semi-conservative (Y/F→L), non-conservative (Y/F→N) or alanine (Y/F/R→A) substitutions. Previously reported gain-of-function (KYKFF) and truncated ‘MDTCS’ variants were also included. Sera from 18 patients were screened against all variants. Conservative mutations of the aromatic residues did not reduce the binding of autoantibodies. Moderate resistance was achieved by replacing R568 and R660 by lysines or alanines. Semi-conservative mutations of aromatic residues showed a moderate effectiveness in autoantibody resistance. Non-conservative asparagine or alanine mutations of aromatic residues were the most effective. In the mixtures of autoantibodies from the majority (89%) of patients screened, autoantibodies targeting the spacer RFRYY epitope were preponderant compared to other epitopes. Reductions in ADAMTS13 proteolytic activity were observed for all full-length mutant variants, in varying degrees. The greatest reductions in activity were observed in the most autoantibodyresistant variants (15-35% residual activity in a FRETS-VWF73 assay). Among these, a triple-alanine mutant . RARAA . showed activity in a von Willebrand factor multimer assay. This study shows that non-conservative and alanine modifications of residues within the exosite-3 spacer RFRYY epitope in full-length ADAMTS13 resist the binding of autoantibodies from patients with immune thrombotic thrombocytopenic purpura, while retaining residual proteolytic activity. Our study provides a framework for the design of autoantibody-resistant ADAMTS13 variants for further therapeutic development.

Destabilization of UHT milk by protease AprX from Pseudomonas fluorescens and plasmin

Destabilization of UHT milk during its shelf life is mainly promoted by the residual proteolytic activity attributed to the psychrotrophic bacterial proteases and native milk proteases. In this study, we built skim UHT milk-based model systems to which either the major bacterial protease (AprX from Pseudomonas fluorescens), or the major native milk protease (plasmin) was added, to allow a direct comparison between the destabilization of skim UHT milk by both categories of enzymes. The physical and chemical properties were studied during 6 weeks. Our results showed AprX induced compact gels when almost all the κ-casein was hydrolyzed and the degree of hydrolysis (DH) exceeded 1.3%. Plasmin induced soft gels when around 60% of both β- and αs1-casein were hydrolyzed and the DH reached 2.1%. The knowledge gained from this study may be used for developing diagnostic tests for determining the protease responsible for UHT milk destabilisation.

Proteolysis of casein micelles by heat-stable protease secreted by Serratia liquefaciens leads to the destabilisation of UHT milk during its storage

Serratia liquefaciens is a psychrotrophic species, frequently found in raw milk, which secretes Ser2, a heat-resistant protease. Involvement of this species in UHT milk destabilisation was investigated in the present study. Microfiltered milk was inoculated independently with strains S. liquefaciens L53 or L64. Then, UHT treatment was performed and stability of the corresponding UHT milk was investigated during three months of storage. The residual proteolytic activity of strain L53 led to destabilisation of UHT milk, with sedimentation and formation of aggregates. Hydrolysis of casein micelles was confirmed by the increase in the content of non-casein nitrogen and the identification of numerous peptides coming from the four caseins using mass spectrometry. For strain L64, no visual and biochemical alteration were found. This study showed that Ser2 resists UHT treatment and could be a cause of UHT milk destabilisation; however, this destabilisation by S. liquefaciens was strain-dependent.

Growth of Pseudomonas weihenstephanensis, Pseudomonas proteolytica and Pseudomonas sp. in raw milk: Impact of residual heat-stable enzyme activity on stability of UHT milk during shelf-life

One of the reasons for spoilage of UHT milk during shelf-life is the presence of residual proteolytic activity produced from Pseudomonas spp. during storage of raw milk. The aim of this study was to describe the product defects occurring in indirectly heated UHT milk during shelf-life, and to establish a correlation between proteolytic activity and onset of product spoilage. UHT milk was produced from raw milk incubated with different Pseudomonas strains, and examined over four months during storage at 20 °C. Inactivation kinetics of the peptidases were determined. In UHT milk, product defects occurred in the order: bitterness – particles – creaming – sediment – gelation in all the samples containing peptidases (apparent enzyme activity ≥ 0.03 pkat mL−1). A linear correlation was found between proteolytic activity and onset of product defects, apart from onset of gelation.

Production of a Milk-Clotting Enzyme by Glutinous Rice Fermentation and Partial Characterization of the Enzyme

Fermentation parameters affecting a milk-clotting enzyme (MCE) production by glutinous rice fermentation were evaluated using selected rice starters called Jiuqu. The MCE was produced under the following conditions: rice inoculation starter at 3%, fermentation time of 5 days, temperature at 30C, initial rice medium pH of 5.0, rice concentration of 70% and rotation speed of 120 rpm. The enzyme preparation obtained by 70% ethanol precipitation of the culture supernatant had milk-clotting activity (MCA) of 6,160 Su/g and a C/P ratio (the ratio between MCA and proteolytic activity) of 10.81 at a recovery rate of 44.82%. The molecular mass of the MCE was 39.7 kDa. MCE production was optimal at 35C and a pH of 5.4. The presence of Na+, K+, Mg2+, Ca2+ and Al3+ stimulated enzyme activity. The Km and Vmax values were 22.90 g/L and 21.11 mL/s, respectively, with skim milk as the substrate. Practical Applications In the last two decades, different sources of milk-clotting enzymes (MCEs) have been explored to meet the increasing demand for cheese production worldwide. This study describes a MCE isolated by ethanol fractionation from glutinous rice wine fermented by Jiuqu. The MCE, with a relatively high C/P ratio, is potentially applicable as a coagulant in cheese making. The property of thermal instability of the enzyme is advantageous as it would be inactivated during curd scalding (at about 50C), thus formation of bitter-taste peptides caused by residual proteolytic activity of the enzyme during cheese ripening could be prevented. In addition, the optimal temperature (35C) of the MCE is within the range of temperature (30–35C) generally used during milk coagulation processes. Therefore, the results suggest that a MCE that could have potential application in cheese production may be obtained by glutinous rice fermentation under the conditions of this study.

Hypochlorous Acid Generated by Neutrophils Inactivates ADAMTS13: AN OXIDATIVE MECHANISM FOR REGULATING ADAMTS13 PROTEOLYTIC ACTIVITY DURING INFLAMMATION

ADAMTS13 is a plasma metalloproteinase that cleaves large multimeric forms of von Willebrand factor (VWF) to smaller, less adhesive forms. ADAMTS13 activity is reduced in systemic inflammatory syndromes, but the cause is unknown. Here, we examined whether neutrophil-derived oxidants can regulate ADAMTS13 activity. We exposed ADAMTS13 to hypochlorous acid (HOCl), produced by a myeloperoxidase-H2O2-Cl(-) system, and determined its residual proteolytic activity using both a VWF A2 peptide substrate and multimeric plasma VWF. Treatment with 25 nm myeloperoxidase plus 50 μm H2O2 reduced ADAMTS13 activity by >85%. Using mass spectrometry, we demonstrated that Met(249), Met(331), and Met(496) in important functional domains of ADAMTS13 were oxidized to methionine sulfoxide in an HOCl concentration-dependent manner. The loss of enzyme activity correlated with the extent of oxidation of these residues. These Met residues were also oxidized in ADAMTS13 exposed to activated human neutrophils, accompanied by reduced enzyme activity. ADAMTS13 treated with either neutrophil elastase or plasmin was inhibited to a lesser extent, especially in the presence of plasma. These observations suggest that oxidation could be an important mechanism for ADAMTS13 inactivation during inflammation and contribute to the prothrombotic tendency associated with inflammation.

A3 Domain Region 1803–1818 Contributes to the Stability of Activated Factor VIII and Includes a Binding Site for Activated Factor IX

A recent chemical footprinting study in our laboratory suggested that region 1803-1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems incompatible with a role of the amino acids 1803-1818 for FIXa or A2 domain binding. In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, and two FVIII variants in which residues 1803-1810 and 1811-1818 are replaced by the corresponding residues of factor V (FV). Enzyme kinetic studies showed that only FVIII/FV 1811-1818 has a decreased apparent binding affinity for FIXa. Flow cytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII/FV 1811-1818 on lipospheres. Site-directed mutagenesis revealed that Phe-1816 contributes to the interaction with FIXa. To evaluate FVIIIa stability, the FVIII/FV chimeras were activated by thrombin, and the decline in cofactor function was followed over time. FVIII/FV 1803-1810 and FVIII/FV 1811-1818 but not FVIII-N1810C showed a decreased FVIIIa half-life. However, when the FVIII variants were activated in presence of FIXa, only FVIII/FV 1811-1818 demonstrated an enhanced decline in cofactor function. Surface plasmon resonance analysis revealed that the FVIII variants K1813A/K1818A, E1811A, and F1816A exhibit enhanced dissociation after activation. The results together demonstrate that the glycan at 1810 is not involved in FVIII cofactor function, and that Phe-1816 of region 1811-1818 contributes to FIXa binding. Both regions 1803-1810 and 1811-1818 contribute to FVIIIa stability.

Comparative study of proteins recovered from whole North Pacific krill Euphausia pacifica by acidic and alkaline treatment during isoelectric solubilization/precipitation

North Pacific krill is widely distributed in the waters around Japan, however its utilization for human consumption has been limited due to its small size. We have investigated the proximate composition, the amino acid, free fatty acid, mineral and protein composition, respectively, as well as the residual proteolytic activity of krill protein, as recovered by acidic- and alkaline-aided isoelectric solubilization/precipitation, in order to facilitate effective utilization of this krill species. Krill protein yield was 31.7 and 73.4 % by acidic and alkaline treatment, respectively. Both treatments resulted in a comparable higher content of essential amino acids than that in whole krill, accompanied with effective removal of insoluble materials. Three major proteolytic active bands with molecular weights of approximately 28, 18 and 16 kDa, as estimated by gelatin-based zymography, were detected. Pronounced residual proteolytic activity was observed in the krill protein after alkaline processing, implying the possible involvement of proteinase(s) in protein degradation during alkaline solubilization. Based on these results, we suggest that inhibition of proteolysis during isoelectric solubilization/precipitation is required for the effective use of North Pacific krill.

Proteolysis of ultra high temperature-treated casein micelles by AprX enzyme from Pseudomonas fluorescens F induces their destabilisation

Destabilisation of ultra high temperature (UHT) treated milk has been linked to residual proteolytic activity after UHT treatment. To understand the physico-chemical modifications of casein micelles by the protease AprX, produced by Pseudomonas fluorescens F, this enzyme was purified and added to raw milk before UHT treatment. Destabilisation of the UHT milk, over three months of storage, was investigated at macroscopic, colloidal and molecular scales. A visual destabilisation appeared progressively over time. At colloidal scale, aggregates were formed and a parallel decrease in zeta potential and hydration of casein micelles was observed. At molecular scale, peptides were released from casein micelles and identified by reversed-phase liquid chromatography coupled with tandem mass spectrometry. The αS1-, αS2-, β- and κ-caseins were hydrolysed, with a preference for β-casein. The results were consistent with the proposition that proteolysis by Ps. fluorescens leading to the destabilisation of milk was due to the activity of AprX.

Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution

Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (K(cat)) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5-fold increased specific activity (15°C) and over 100 times prolonged half-life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity-altering amino acid substitutions were from non-charged to non-charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions.

Human ZMPSTE24 disease mutations: residual proteolytic activity correlates with disease severity

The zinc metalloprotease ZMPSTE24 plays a critical role in nuclear lamin biology by cleaving the prenylated and carboxylmethylated 15-amino acid tail from the C-terminus of prelamin A to yield mature lamin A. A defect in this proteolytic event, caused by a mutation in the lamin A gene (LMNA) that eliminates the ZMPSTE24 cleavage site, underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Likewise, mutations in the ZMPSTE24 gene that result in decreased enzyme function cause a spectrum of diseases that share certain features of premature aging. Twenty human ZMPSTE24 alleles have been identified that are associated with three disease categories of increasing severity: mandibuloacral dysplasia type B (MAD-B), severe progeria (atypical 'HGPS') and restrictive dermopathy (RD). To determine whether a correlation exists between decreasing ZMPSTE24 protease activity and increasing disease severity, we expressed mutant alleles of ZMPSTE24 in yeast and optimized in vivo yeast mating assays to directly compare the activity of alleles associated with each disease category. We also measured the activity of yeast crude membranes containing the ZMPSTE24 mutant proteins in vitro. We determined that, in general, the residual activity of ZMPSTE24 patient alleles correlates with disease severity. Complete loss-of-function alleles are associated with RD, whereas retention of partial, measureable activity results in MAD-B or severe progeria. Importantly, our assays can discriminate small differences in activity among the mutants, confirming that the methods presented here will be useful for characterizing any new ZMPSTE24 mutations that are discovered.

Investigation of the structure and proteolytic activity of papain in aqueous miscible organic media

The stability of papain was studied in aqueous–organic mixtures by means of residual proteolytic activity along with various spectroscopic analyses (fluorescence and ATR-FTIR combined with isotopic exchange with D 2O). The investigated systems contained 1 or 10% (v/v) of an aqueous buffered solution (pH 8.0) in acetonitrile (ACN), methanol (MeOH) or dimethyl formamide (DMF). The results evidenced that papain retained almost all its catalytic activity after 24 h of incubation in the presence of ACN, and a more compact conformation of the enzyme was detected. Papain suffered an important loss of enzymatic activity (ca. 80%) after 24 h incubation in MeOH although, no global conformational change and minor secondary structure rearrangements were detected. This observation suggests that somehow the active site region was altered. On the other hand, papain suffered a complete inactivation when exposed to those media containing DMF. Fluorescence analyses revealed that an irreversible conformational change took place after 24 h incubation, and a moderate increase in β-sheet and β-turn structures was the most relevant finding when secondary structure was analyzed. The evidences demonstrated that the organic solvents induce a more rigid and compact structure of papain regardless of the organic:buffer ratio investigated. In turn, these modifications affect the active catalytic site in the particular case of MeOH and DMF. These findings were in agreement with the thermo-stability of the enzyme performed after heating at 353 K in all the studied media, that is the presence of ACN did not substantially affect the secondary structure of papain. Nevertheless, the α-helix domain demonstrated to be less thermally stable than the β-sheet domain, turning into aggregated structures after heating, especially in the presence of MeOH and DMF.

Fragmentation of a highly purified monoclonal antibody attributed to residual CHO cell protease activity

Monoclonal antibody (mAb) fragmentation can be a widespread problem across the biotechnology industry and there is a current need to better understand the underlying principles. Here, we report an example of a high-purity human IgG1 mAb prepared from CHO cells exhibiting fragmentation that can be attributed to residual proteolytic enzyme activity. The concomitant occurrence of proteolytic and non-proteolytic peptide bond cleavage is shown and the respective fragmentation patterns characterized using high-resolution LC-MS. Fragmentation rates are monitored by SE-HPLC and SDS-PAGE over the pH range 4-6 and characterized in the presence and absence of pepstatin A, an inhibitor of acidic proteases. After 20 days at 40°C, pH 4, ∼60% decrease in BIIB-mAb monomer peak occurred attributed to residual proteolytic activity. At pH 5, this value was ∼13%. These results have implications for formulation design studies and the interpretation of accelerated stability data. A simple method to screen for acidic protease activity using the proteolytic enzyme inhibitor pepstatin A is described.