Disulfide Cleavage Research Articles

Porous silicon surface modification via a microwave-induced in situ cyclic disulfide (S-S) cleavage and Si-S bond formation

Porous silicon (pSi) materials have gained a great deal of attention from various research fields, and their surface-functionalization is one of the critical points for their applications. In this study, a new surface modification method of Si-H-terminated pSi materials via microwave-induced Si-S bond formation is disclosed. The silicon hydride (Si−H) functionality on the pSi surface could react with the 5-membered cyclic disulfide (S-S) compound (DL-α-lipoic acid in this study) by microwave-induced in situ S-S bond cleavage and Si-S bond formation. This surface chemistry is fast responsive (<10 min) and more efficient than other methods such as vortexing, heating stirring, or ultrasonication. The reaction maintains the primary porous structure of pSi materials including pSi wafer, pSi rugate filer, and pSi nanoparticles. An additional functional group such as carboxylic acid is demonstrated to be readily introducible on the pSi surface for further applications. Overall, this study has successfully demonstrated the porous silicon surface modification via a microwave-induced in situ cyclic disulfide (S-S) cleavage and Si-S bond formation.

Structure of a dinickel(II)-dithiolate bridged macrocyclic complex synthesised via a novel solvent-assisted disulfide cleavage reaction

The reaction of 2,6-diformyl-4-methylphenyl disulfide with [Ni(tn)3]Cl2 (tn = 1,3-diaminopropane), in methanol and in the presence of NaBPh4 resulted in the isolation of [Ni2(L1)](DMF)2(BPh4)2 (I), (L1 = 15,95-dimethyl-3,7,11,15-tetraaza-1,9(1,3)-dibenzenacyclohexadecaphane-2,7,10,15-tetraene-12,92-bis(thiolate). This reaction represents a 2-electron solvent assisted reduction of the disulfide bond to form two thiolate ligands where methanol is converted to formaldehyde and the two nickel centres remained in the + 2 oxidation state. The crystal structure of I showed the two nickel(II) atoms are bridged by two thiolates in a binuclear Ni2S2 core inside of a macrocyclic framework. The IR spectrum showed a band at 1625 cm−1, assigned to the imine ν(C N) stretch of the macrocyclic ligand, and bands at 734, 706 cm−1, are assigned to the tetraphenylborate counter ions. This is the first example of solvent assisted SS bond cleavage being used to prepare a dicompartmental macrocyclic complex.

Disulfide cleavage to improve interfacial behavior and emulsification properties of oat protein

Disulfide bonds play an essential structural role but may hinder the molecular flexibility and functionality of proteins. The present study investigated the effect of disulfide cleavage on emulsifying activity of oat protein isolate (OPI). Four reducing agents tested (dithiothreitol, ascorbic acid, cysteine, and sodium bisulfite) except ascorbic acid disrupted inter-subunit SS bonds of OPI (up to 90 %) in a dose-dependent manner. Emulsification properties were measured specifically on cysteine-modified OPI, and the results showed increased emulsifying activity up to 37 % after subunit dissociation, which exposed hydrophobic groups and loosened the structure. In particular, emulsions formed by cysteine-treated OPI (1.7 to 6.7 mM/mg protein) displayed a superior interfacial protein coverage (0.170 m2/mg compared to 0.092 m2/mg for control) and reduced emulsion particle size (from 4722 to 2238 nm). The application of cysteine as a structure-modifying food additive can broaden the utilization of oat protein in emulsion-based food products.

Near UV and Visible Light Photo-Degradation Mechanisms in Citrate Buffer: One-Electron Reduction of Peptide and Protein Disulfides promotes Oxidation and Cis/Trans Isomerization of Unsaturated Fatty Acids of Polysorbate 80

Citrate is a common buffer for slightly acidic pH ranges of protein formulations. In the presence of iron, citrate buffer undergoes photo-degradation induced by near UV and visible light. Recent studies (Subelzu and Schöneich, Mol. Pharm. 2020, 17, 4163-4179) have documented that such photo-degradation results in the formation of carbon dioxide radical anion (•CO2−), a strong reductant which reduces Fe3+, O2, and disulfide bonds. In the present study we show that near UV and visible light photo-degradation of citrate in the presence of iron can induce reductive peptide and protein disulfide cleavage as well as free radical damage of a surfactant, polysorbate 80 (PS80). Reductive disulfide cleavage is most likely caused by efficient electron transfer from carbon dioxide radical anions to disulfides, resulting in the generation of thiol/thiolate and thiyl radicals. The latter can react with mono- and polyunsaturated fatty acids of PS80 to cause cis/trans isomerization and/or oxidation. Representative products generated by cis/trans isomerization and oxidation of oleic acid esters have been detected by HPLC-MS analysis. Further evidence for reductive disulfide cleavage was obtained through the analysis of free thiols. The oxidation of PS80 can also be the consequence of reactive oxygen species (ROS) generation through the reduction of O2 by carbon dioxide radical anions and/or intermediary Fe2+.

Structure of a Dinickel(Ii)-Dithiolate Bridged Macrocyclic Complex Synthesised Via a Novel Solvent-Assisted Disulfide Cleavage Reaction

Structure of a Dinickel(Ii)-Dithiolate Bridged Macrocyclic Complex Synthesised Via a Novel Solvent-Assisted Disulfide Cleavage Reaction

Rapid Disulfide Mapping in Peptides and Proteins by meta-Chloroperoxybenzoic Acid (mCPBA) Oxidation and Tandem Mass Spectrometry.

Disulfide bonds are a class of important post-translational modifications that play important roles in modulating the structures and functions of proteins. Therefore, the mapping of disulfide linkages in peptides and proteins is indispensable for complete structure characterization and functional studies. As disulfide bonds in protonated ions do not dissociate readily under low-energy collision-induced dissociation (CID), they are usually chemically cleaved or activated prior to mass spectrometry (MS) or tandem MS (MS/MS) analysis. In this study, we report a new method that allows the mapping of disulfide linkages in peptides and proteins through meta-chloroperoxybenzoic acid (mCPBA)-based disulfide oxidation and MS/MS. Upon oxidation, the disulfide bond is converted to a thiosulfinate group, i.e., S(═O)-S, in a rapid (>60% yield in 1 min) and highly specific approach in an aqueous phase. The thiosulfinate group is then preferentially cleaved by MS/MS. For interchain disulfide linkages, this leads to a facile peptide chain separation and the identification of disulfide-linked peptides. For intrachain disulfide linkages, collisional activation of the thiosulfinate leads to disulfide cleavage and fragmentation of the peptide backbone constrained by the disulfide loop, enabling a near-complete peptide sequencing. The mCPBA oxidation-based disulfide mapping strategy can be readily integrated with bottom-up or top-down protein analysis for comprehensive protein structure elucidation, e.g., digested lysozyme and intact human insulin.

Exceptionally rich keratinolytic enzyme profile found in the rare actinomycetes Amycolatopsis keratiniphila D2T.

The non-spore forming Gram-positive actinomycetes Amycolatopsis keratiniphila subsp. keratiniphila D2T (DSM 44,409) has a high potential for keratin valorization as demonstrated by a novel biotechnological microbial conversion process consisting of a bacterial growth phase and a keratinolytic phase, respectively. Compared to the most gifted keratinolytic Bacillus species, a very large number of 621 putative proteases are encoded by the genome of Amycolatopsis keratiniphila subsp. keratiniphila D2T, as predicted by using Peptide Pattern Recognition (PPR) analysis. Proteome analysis by using LC-MS/MS on aliquots of the supernatant of A. keratiniphila subsp. keratiniphila D2T culture on slaughterhouse pig bristle meal, removed at 24, 48, 96 and 120h of growth, identified 43 proteases. This was supplemented by proteome analysis of specific fractions after enrichment of the supernatant by anion exchange chromatography leading to identification of 50 proteases. Overall 57 different proteases were identified corresponding to 30% of the 186 proteins identified from the culture supernatant and distributed as 17 metalloproteases from 11 families, including an M36 protease, 38 serine proteases from 4 families, and 13 proteolytic enzymes from other families. Notably, M36 keratinolytic proteases are prominent in fungi, but seem not to have been discovered in bacteria previously. Two S01 family peptidases, named T- and C-like proteases, prominent in the culture supernatant, were purified and shown to possess a high azo-keratin/azo-casein hydrolytic activity ratio. The C-like protease revealed excellent thermostability, giving promise for successful applications in biorefinery processes. Notably, the bacterium seems not to secrete enzymes for cleavage of disulfides in the keratinous substrates. KEY POINTS: • A. keratiniphila subsp. keratiniphila D2T is predicted to encode 621 proteases. • This actinomyceteefficiently converts bristle meal to a protein hydrolysate. • Proteome analysis identified 57 proteases in its secretome.

Camptothecin Nanoprodrug Possessing Dual Responsiveness to Endolysosomal pH and Cytosolic Redox for Amplified Cytotoxic Potency.

Selective activation of prodrug nanomedicine in cell interiors is deemed to be crucial in pursuit of precision anti-tumor therapy. In the present study, we attempted to synthesize an amphiphilic diblock copolymer poly(ethylene glycol)-polylysine (PEG-PLys) based on ring-opening polymerization. The γ terminal amines of lysine units were conjugated with camptothecin (CPT) through redox-responsive disulfide linkage, followed by conversion of the rest of the amines of PLys into carboxyl groups. Core-shell architectural nanoparticles could be achieved by self-assembly of the yielded amphiphiles characterized to possess CPT-linked PLys segments as the internal core and PEG segments as the external shell. Furthermore, attempts were made to precipitate CaPO3 on the yielded core with the aid of the carboxyl groups. Subsequent investigations confirmed uniform nanoscale formation with a hydrodynamic diameter of approximately 63.0 nm and excellent colloidal stabilities. Most importantly, the proposed dually responsive prodrug construct was determined to possess intriguing sequentially intracellular microenvironment-responsive functionalities: (1) the inorganic CaPO3 precipitate could not only exclude the internal payloads from premature reactions but also rapidly dissolve in acidic endosomal compartments, with the induced osmotic pressure thereby facilitating translocation of the prodrug into the cytosol; (2) CPT could be readily metabolized due to disulfide cleavage responsive to the redox potential in cytosolic compartments. Hence, the amalgamated dual-responsiveness eventually contributes to drastic cytotoxic potency, which portends prosperous utilities as precision therapeutics in the treatment of a variety of intractable tumors.

Preparation, characterization, and in vitro drug release behavior of thiolated alginate nanoparticles loaded budesonide as a potential drug delivery system toward inflammatory bowel diseases

For site-specific drug delivery in inflammatory bowel disease, reducible sodium alginate nanoparticles cross-linked with disulfide linkage were developed. Nanoparticles were synthesized in deionized water through self-assembly of amphiphilic thiolated sodium alginate Alg-Cys and subsequently produced cross-linking of disulfide bonds. TEM image showed a spherical core-shell configuration with a size of about 430 nm for the nanoparticles. Dynamic light scattering (DLS) showed high stability, narrow size distribution, and pH-dependent swelling transition for the nanoparticles. Cytotoxicity study showed that there was no evident cell inhibition among the nanoparticles. Also, the size of the nanoparticles increased in 10 mM glutathione (GSH) solution due to the cleavage of disulfides within their network structures. Compared to that in GSH-free buffer, there was a remarkable increase in drug release in pH 7.4 buffer with GSH from drug-loaded nanoparticles, indicating that the nanoparticles could be used for colon-specific drug delivery.

Rhodium-Catalyzed Synthesis of Organosulfur Compounds Involving S-S Bond Cleavage of Disulfides and Sulfur.

Organosulfur compounds are widely used for the manufacture of drugs and materials, and their synthesis in general conventionally employs nucleophilic substitution reactions of thiolate anions formed from thiols and bases. To synthesize advanced functional organosulfur compounds, development of novel synthetic methods is an important task. We have been studying the synthesis of organosulfur compounds by transition-metal catalysis using disulfides and sulfur, which are easier to handle and less odiferous than thiols. In this article, we describe our development that rhodium complexes efficiently catalyze the cleavage of S-S bonds and transfer organothio groups to organic compounds, which provide diverse organosulfur compounds. The synthesis does not require use of bases or organometallic reagents; furthermore, it is reversible, involving chemical equilibria and interconversion reactions.

Conjugation of Doxorubicin to siRNA Through Disulfide-based Self-immolative Linkers.

Co-delivery systems of siRNA and chemotherapeutic drugs have been developed as an attractive strategy to optimize the efficacy of chemotherapy towards cancer cells with multidrug resistance. In these typical systems, siRNAs are usually associated to drugs within a carrier but without covalent interactions with the risk of a premature release and degradation of the drugs inside the cells. To address this issue, we propose a covalent approach to co-deliver a siRNA-drug conjugate with a redox-responsive self-immolative linker prone to intracellular glutathione-mediated disulfide cleavage. Herein, we report the use of two disulfide bonds connected by a pentane spacer or a p-xylene spacer as self-immolative linker between the primary amine of the anticancer drug doxorubicin (Dox) and the 2′-position of one or two ribonucleotides in RNA. Five Dox-RNA conjugates were successfully synthesized using two successive thiol-disulfide exchange reactions. The Dox-RNA conjugates were annealed with their complementary strands and the duplexes were shown to form an A-helix sufficiently stable under physiological conditions. The enzymatic stability of Dox-siRNAs in human serum was enhanced compared to the unmodified siRNA, especially when two Dox are attached to siRNA. The release of native Dox and RNA from the bioconjugate was demonstrated under reducing conditions suggesting efficient linker disintegration. These results demonstrate the feasibility of making siRNA-drug conjugates via disulfide-based self-immolative linkers for potential therapeutic applications.

Cleavage of Disulfide Bonds in Cystine by UV-B Illumination Mediated by Tryptophan or Tyrosine as Photosensitizers.

Photolytic cleavage of disulfide bonds in proteins by UV light will influence their structure and functionality. The present study aimed to investigate the efficiency of disulfide cleavage by UV-B light in a system without a protein backbone consisting of combinations of cystine (a disulfide) and tryptophan (Trp) or tyrosine (Tyr) under anaerobic and aerobic conditions and to identify oxidation products formed by UV-B light. Cystine was reduced to cysteine (Cys) almost with a 1:1 stoichiometry by photoexcited Trp for anaerobic equimolar aqueous solutions (each 200 μM; pH 7.0), while photoexcited Tyr provided lower concentrations of Cys. The calculation of apparent quantum yields allowed for a comparison between the efficiency of reactions and showed that formation of Cys from disulfide cleavage of cystine was more efficient by photoexcited Trp than by photoexcited Tyr and of cystine alone and that Trp was more sensitive to photodegradation than Tyr and cystine under both aerobic and anaerobic conditions. Increasing the ratio between cystine and Trp to a 1:2 ratio did not increase the efficiency of free thiol formation but caused a more efficient photodegradation of Trp. The free thiol formed from disulfide cleavage of cystine was further oxidized to other unidentified compounds. Trp oxidation products (3-hydroxykynurenine (3-OH-Kyn) and tryptamine) were only identified in minor concentrations following light exposure of cystine and Trp in 1:1 and 1:2 ratios under both aerobic and anaerobic conditions, indicating further photodegradation to unidentified compounds. 3,4-Dihydroxyphenylalanine (DOPA) was formed from the oxidation of Tyr in the illuminated samples of cystine and Tyr in a 1:1 ratio under both aerobic and anaerobic conditions.

A glutathione-responsive photosensitizer with fluorescence resonance energy transfer characteristics for imaging-guided targeting photodynamic therapy

Here, we have synthesized and characterized a novel activatable photosensitizer (PS) 8a in which two well-designed boron dipyrromethene (BODIPY) derivatives are utilized as the photosensitizing fluorophore and quencher respectively, which are connected by a disulfide linker via two successive Cu (І) catalyzed click reactions. The fluorescence emission and singlet oxygen production of 8a are suppressed via intramolecular fluorescence resonance energy transfer (FRET) from the excited BODIPY-based PS part to quencher unit, but both of them can be simultaneously switched on by cancer-related biothiol glutathione (GSH) in phosphate buffered saline (PBS) solution with 0.05% Tween 80 as a result of cleavage of disulfide. Also, 8a exhibits a bright fluorescence image and a substantial ROS production in A549 human lung adenocarcinoma, HeLa human cervical carcinoma and H22 mouse hepatoma cells having a relatively high concentration of GSH, thereby leading to a significant photocytotoxicity, with IC50 values as low as 0.44 μM, 0.67 μM and 0.48 μM, respectively. In addition, the photosensitizer can be effectively activated and imaged in H22 transplanted hepatoma tumors of mice and shows a strong inhibition on tumor growth. All these results suggest that such a GSH-responsive photosensitizer based on FRET mechanism may provide a new strategy for tumor-targeted and fluorescence imaging-guided cancer therapy.

Disulfide-Based Self-Immolative Linkers and Functional Bioconjugates for Biological Applications.

It is of vital importance to reversibly mask and selectively activate bioactive agents for advanced therapeutic and diagnostic purposes, aiming to efficiently suppress background interferences and attenuate systemic toxicity. This strategy has been involved in diverse applications spanning from chemical/biological sensors and diagnostics to drug delivery nanocarriers. Among these, redox-responsive disulfide linkages have been extensively utilized by taking advantage of extracellular and intracellular glutathione (GSH) gradients. However, direct conjugation of cleavable triggers to bioactive agents through disulfide bonds suffers from bulky steric hindrance and limited choice of trigger-drug combinations. Fortunately, the emergence of disulfide self-immolative linkers (DSILs) provides a general and robust strategy to not only mask various bioactive agents through the formation of dynamic disulfide linkages but also make it possible to be selectively activated upon disulfide cleavage in the reductive cytoplasmic milieu. In this review, recent developments in DSILs are focused with special attention on emerging chemical design strategies and functional applications in the biomedical field.

The effect of myeloperoxidase isoforms on biophysical properties of red blood cells.

Myeloperoxidase (MPO), an oxidant-producing enzyme, stored in azurophilic granules of neutrophils has been recently shown to influence red blood cell (RBC) deformability leading to abnormalities in blood microcirculation. Native MPO is a homodimer, consisting of two identical protomers (monomeric MPO) connected by a single disulfide bond but in inflammatory foci as a result of disulfide cleavage monomeric MPO (hemi-MPO) can also be produced. This study investigated if two MPO isoforms have distinct effects on biophysical properties of RBCs. We have found that hemi-MPO, as well as the dimeric form, bind to the glycophorins A/B and band 3protein on RBC's plasma membrane, that lead to reduced cell resistance to osmotic and acidic hemolysis, reduction in cell elasticity, significant changes in cell volume, morphology, and the conductance of RBC plasma membrane ion channels. Furthermore, we have shown for the first time that both dimeric and hemi-MPO lead to phosphatidylserine (PS) exposure on the outer leaflet of RBC membrane. However, the effects of hemi-MPO on the structural and functional properties of RBCs were lower compared to those of dimeric MPO. These findings suggest that the ability of MPO protein to influence RBC's biophysical properties depends on its conformation (dimeric or monomeric isoform). It is intriguing to speculate that hemi-MPO appearance in blood during inflammation can serve as a regulatory mechanism addressed to reduce abnormalities on RBC response, induced by dimeric MPO.

Development of a Cysteine-Conjugatable Disulfide FRET Probe: Influence of Charge on Linker Cleavage and Payload Trafficking for an Anti-HER2 Antibody Conjugate.

Disulfide-linked bioconjugates allow the delivery of pharmacologically active or other cargo to specific tissues in a redox-sensitive fashion. However, an understanding of the kinetics, subcellular distribution, and mechanism of disulfide cleavage in such bioconjugates is generally lacking. Here, we report a modular disulfide-linked TAMRA-BODIPY based FRET probe that can be readily synthesized, modified, and conjugated to a cysteine-containing biomolecule to enable real-time monitoring of disulfide cleavage during receptor-mediated endocytosis in cells. We demonstrate the utility of this probe to study disulfide reduction during HER2 receptor-mediated uptake of a Cys-engineered anti-HER2 THIOMAB antibody. We found that introduction of positive, but not negative, charges in the probe improved retention of the BODIPY catabolite. This permitted the observation of significant disulfide cleavage in endosomes or lysosomes on par with proteolytic cleavage of a similarly charged valine-citrulline peptide-based probe. In general, the FRET probe we describe should enable real-time cellular monitoring of disulfide cleavage in other targeted delivery systems for mechanistic or diagnostic applications. Furthermore, modifications to the released BODIPY moiety permit evaluation of physicochemical properties that govern lysosomal egress or retention, which may have implications for the development of next-generation antibody-drug conjugates.

Quantification of Aldehydes on Polymeric Microbead Surfaces via Catch and Release of Reporter Chromophores.

Aldehyde moieties on 2D-supports or micro- and nanoparticles can function as anchor groups for the attachment of biomolecules or as reversible binding sites for proteins on cell surfaces. The use of aldehyde-based materials in bioanalytical and medical settings calls for reliable methods to detect and quantify this functionality. We report here on a versatile concept to quantify the accessible aldehyde moieties on particle surfaces through the specific binding and subsequent release of small reporter molecules such as fluorescent dyes and nonfluorescent chromophores utilizing acylhydrazone formation as a reversible covalent labeling strategy. This is representatively demonstrated for a set of polymer microparticles with different aldehyde labeling densities. Excess reporter molecules can be easily removed by washing, eliminating inaccuracies caused by unspecific adsorption to hydrophobic surfaces. Cleavage of hydrazones at acidic pH assisted by a carbonyl trap releases the fluorescent reporters rapidly and quasi-quantitatively and allows for their fluorometric detection at low concentration. Importantly, this strategy separates the signal-generating molecules from the bead surface. This circumvents common issues associated with light scattering and signal distortions that are caused by binding-induced changes in reporter fluorescence as well as quenching dye-dye interactions on crowded particle surfaces. In addition, we demonstrate that the release of a nonfluorescent chromophore via disulfide cleavage and subsequent quantification by absorption spectroscopy gives comparable results, verifying that both assays are capable of rapid and sensitive quantification of aldehydes on microbead surfaces. These strategies enable a quantitative comparison of bead batches with different functionalization densities, and a qualitative prediction of their coupling efficiencies in bioconjugations, as demonstrated in reductive amination reactions with Streptavidin.

Catalytic Cleavage of Disulfide Bonds in Small Molecules and Linkers of Antibody-Drug Conjugates.

In cells, catalytic disulfide cleavage is an essential mechanism in protein folding and synthesis. However, detailed enzymatic catalytic mechanism relating cleavage of disulfide bonds in xenobiotics is not well understood. This study reports an enzymatic mechanism of cleavage of disulfide bonds in xenobiotic small molecules and antibody conjugate (ADC) linkers. The chemically stable disulfide bonds in substituted disulfide-containing pyrrolobenzodiazepine (PBD, pyrrolo[2,1-c][1,4]benzodiazepine) monomer prodrugs in presence of glutathione or cysteine were found to be unstable in incubations in whole blood of humans and rats. It was shown the enzymes involved were thioredoxin (TRX) and glutaredoxin (GRX). For a diverse set of drug-linker conjugates, we determined that TRX in the presence of TRX-reductase and NADPH generated the cleaved products that are consistent with catalytic disulfide cleavage and linker immolation. GRX was less rigorously studied; in the set of compounds studied, its role in the catalytic cleavage was also confirmed. Collectively, these in vitro experiments demonstrate that TRX as well as GRX can catalyze the cleavage of disulfide bonds in both small molecules and linkers of ADCs.

Enhanced Aggregability of AIE-Based Probe through H2S-Selective Triggered Dimerization and Its Applications to Biological Systems

We developed a disulfide-conjugated aggregation-induced emission luminogens (AIEgen) (1) that can dramatically enhance the aggregability through a H2S-selective disulfide cleavage and its dimerizat...

Degradable Protein-loaded Polymer Capsules Fabricated by Thiol-disulfide Cross-linking Reaction at Liquid-liquid Interface

In these years, the encapsulation of proteins for protection and delivery purpose has attracted great interest. In this research, W/O emulsion droplets were used as soft templates and bovine serum albumin (BSA) encapsulated hollow capsules were prepared by liquid-liquid interfacial thiol-disulfide exchange reaction. Block copolymer chains with pendant pyridyl disulfide groups are located at liquid-liquid interface, and upon addition of a macromolecular crosslinking agent with multiple pendant thiol groups into an emulsion, thiol-disulfide interfacial crosslinking reactions lead to the formation of BSA encapsulated hollow capsules. The cleavage of disulfides on the membranes results in the degradation of hollow structures and the release of encapsulated protein molecules. Transmission electron microscopy, scanning electron microscopy, atomic force microscopy, and confocal laser scanning microscopy were employed to characterize the hollow capsules. In comparison with native BSA, BSA molecules encapsulated in the hollow structures show higher catalytic efficiency due to higher local concentration of reactants in the structures. The membranes of the hollow capsules can efficiently protect the encapsulated BSA from hydrolysis by trypsin.