Use of the Computer Automated Structure Evaluation program in determining quantitative structure-activity relationships within hallucinogenic phenylalkylamines

The Computer Automated Structure Evaluation (CASE) program has been successfully used to generate automatically and identify molecular fragments relevant to the hallucinogenic activity expressed by some phenylalkylamines. Utilizing these major fragments, Quantitative Structure-Activity Relationship (QSAR) calculations were carried out to obtain an equation which was used for predictions of potencies. Correlations of these major activating/inactivating fragments with the biological activity of the compounds, as well as predictive capabilities of the CASE program, are discussed.     

Ovostatin: a novel proteinase inhibitor from chicken egg white. II. Mechanism of inhibition studied with collagenase and thermolysin

The inhibition mechanism of ovostatin was studied using rabbit synovial collagenase and thermolysin. When enzymes were complexed with ovostatin, only the proteolytic activity towards high molecular weight substrates was inhibited. Activity towards low molecular weight substrates was partially modified: the catalytic activity of collagenase bound to ovostatin was inhibited by only 40% towards 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg and that of thermolysin bound to ovostatin was activated about 2.6-fold towards benzyloxycarbonyl-Gly-Leu-NH2 and benzyloxycarbonyl-Gly-Phe-NH2. Collagenase-ovostatin complexes failed to react with anti-(collagenase) antibody. Saturation of ovostatin with thermolysin prevented the subsequent binding of collagenase. Ovostatin-proteinase complexes ran faster than free ovostatin on 5% polyacrylamide gel electrophoresis. Complexing ovostatin with either collagenase or thermolysin resulted in the cleavage of the quarter-subunit of ovostatin (Mr = 165,000) into two fragments with Mr = 88,000 and 78,000. On the other hand, when the inhibitory capacity of ovostatin was tested with trypsin, chymotrypsin, and papain, only partial inhibition of their proteolytic activities was observed towards azocasein. Stronger inhibition was noted when Azocoll was a substrate, however. Analyses of ovostatin-enzyme complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the quarter-subunit of ovostatin was cleaved into several fragments by those enzymes. These results led us to propose that ovostatin inhibits metalloproteinases in preference to proteinases of other classes in a manner similar to alpha 2-macroglobulin; hydrolysis of a peptide bond by a proteinase in the susceptible region of the ovostatin polypeptide chain triggers a conformational change in the ovostatin molecule and the enzyme becomes bound to ovostatin in such a way that the proteinase is sterically hindered from access to large protein substrates and yet is accessible to small synthetic substrates. A kinetic study of collagenase binding to ovostatin gave the value of k2/Ki = 6.3 X 10(5) M-1 min-1. The results indicate that ovostatin is equally as good a substrate for collagenase as type I collagens.     

Independent folding of autolytic fragments of thermolysin and their domain-like properties

High molecular weight autolysis products from thermolysin have been isolated and identified. The primary fragments correspond to residues 1 to 187-204 (21kD) and residues 187-204 to 316 (12kD), respectively. The fragments are both capable of independent refolding upon removal of denaturant. On the basis of these results, we suggest that the first step in the unfolding pathway of thermolysin involves unfolding of an interdomain region and domain separation. Bound calcium ions at sites 1, 2 and 4 play a major role in protecting the protein against both autolysis and unfolding, probably by stabilizing the interdomain region and enhancing domain-domain interactions.     

Inhibitory effects of alcohols on thermolysin activity as examined using a fluorescent substrate

Alcohols inhibit the thermolysin-catalyzed hydrolysis of N-[3-(2-furyl)acryloyl]-Gly-L-Leu-NH(2) and decrease the NaCl-induced activation of thermolysin in a concentration-dependent manner [K. Inouye et al. (1997) J. Biochem. 122, 358-364]. In this study, the inhibitory effects of alcohols on thermolysin activity were examined in detail using 10 different alcohols and a fluorescent substrate, (7-methoxycoumarin-4-yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2). The inhibition by all alcohols examined is completely reversible, and thermolysin activity is recovered by dilution. The inhibitor constants (K(i)) are in the range of 35-430 mM, and the order of the inhibitory effect is 1-pentanol, 1-propanol, 2-butanol, 2-methyl-1-propanol > 1-butanol > 2-propanol > ethanol, tert-amyl alcohol > tert-butyl alcohol > methanol. Linear and secondary alcohols whose mains chains consist of more than 3 carbons inhibit thermolysin effectively. Thermolysin activity is decreased by decreasing the dielectric constant, D, of the reaction medium containing the alcohol, and the decrease depending on the D value was almost the same manner for all alcohols except methanol, tert-butyl alcohol, and tert-amyl alcohol. Alcohols may inhibit thermolysin activity both by binding to the active site, most possibly to the S1' subsite, of thermolysin and by altering the electrostatic and hydrophobic environment around the thermolysin molecule.     

Evolution of alpha 2-macroglobulin. The purification and characterization of a protein homologous with human alpha 2-macroglobulin from plaice (Pleuronectes platessa L.) plasma.

A papain-binding protein (PB-protein) was purified to homogeneity from the plasma of plaice (Pleuronectes platessa L.). PB-protein inhibited the activity of trypsin and pancreatic elastase (serine proteinases), thermolysin (a metalloproteinase) and papain (a cysteine proteinase). Presaturation of PB-protein with trypsin prevented the subsequent inhibition of thermolysin, and vice versa. Only catalytically active endopeptidases were bound by PB-protein. The catalytic activity of trypsin bound by PB-protein was inhibited by 95% against an insoluble protein substrate, but only by 38% against a low-molecular-weight synthetic substrate. The remaining activity of the bound trypsin was partially protected against further inhibition by soya-bean trypsin inhibitor. Trypsin bound by PB-protein showed a decrease of 67% in its reactivity with antibodies. The inhibitory activity of PB-protein was inactivated at pH 8.0 by methylamine (0.2M) or dithiothreitol (1 mM). The inhibition of proteinases by plaice PB-protein shows the distinctive characteristics of inhibition by human alpha 2-macroglobulin, and it is concluded that the plaice protein is a homologue of the human macroglobulin.     

Characterization of cleavage products in selected human lutropin preparations. A protease-sensitive site in human lutropin beta subunit

Low molecular weight fragments derived from the beta subunit of human lutropin have been frequently observed. These fragments are detected by polyacrylamide gel electrophoresis in sodium dodecyl sulfate following reduction of the disulfide bonds. A sample of human lutropin was identified that had a major portion of its beta subunit showing this proteolytic nick. Over 83% of the subunit was nicked based on reduction, carboxymethylation, and isolation of the low molecular weight fragments. This preparation had 53% of the activity of an intact human lutropin (radioligand assay). The proteolytic nick in the subunit was shown by N-terminal sequencing of the C-terminal fragments to be derived from three clips in a hexapeptide region (residues 44-49) characterized by hydrophobic alkyl side chains. Specific clips were on the amino side of Leu-45 (8%), Val-48 (45%) and Leu-49 (47%). Thus the proteolytic activity, presumably derived from the pituitary during processing, has a substrate specificity reminiscent of the bacterial protease, thermolysin.     

Folding of thermolysin fragments. Correlation between conformational stability and antigenicity of carboxyl-terminal fragments

Circular dichroism (CD) and immunochemical measurements have been used to examine conformational properties of COOH-terminal fragments 121-316, 206-316 and 225(226)-316 of thermolysin, and to compare these properties to those of native thermolysin and thermolysin S, the stable partially active two-fragment complex composed of fragments 5-224(225) and 225(226)-316. In aqueous solution at neutral pH, all the COOH-terminal fragments attain a native-like conformation, as judged both by the content of secondary structure deduced from far-ultraviolet CD spectra and by the recognition of rabbit polyclonal antibodies specific for the COOH-terminal region in native thermolysin. The three fragments showed reversible cooperative unfolding transitions mediated by both heat and guanidine hydrochloride (Gdn X HCl). The phase transition curves were analyzed for Tm (temperature of half-denaturation) and Gibbs free energies (delta GD) of unfolding from native to denatured state. The observed order of thermal stability is 225(226)-316 less than or equal to 206-316 less than 121-316 less than thermolysin S less than thermolysin. The ranking of delta GD values for the three fragments correlates with the size of each fragment. Competitive binding studies by radioimmunoassay using 14C-labeled thermolysin and affinity purified antibodies specific for native antigenic determinants in segment 206-316 of native thermolysin indicate that the COOH-terminal fragments adopt native-like conformations which are in equilibrium with non-native conformations. These equilibria are shifted towards the native state as the fragment size increases from 225(226)-316, to 206-316, to 121-316. Fragment 225(226)-316, when combined with fragment 5-224(225) in the thermolysin S complex, adopts a more stable native-like conformation and becomes much more antigenic. It has been shown that the degree of antigenicity of COOH-terminal fragments towards thermolysin antibodies correlates directly with their conformational stability. The results of this study are discussed in relation to the recently proposed correlation between antigenicity and segmental mobility of globular proteins.     

Alternative model for the internal structure of laminin

A monoclonal antibody to laminin, LMN-1, was generated by immunizing rats with laminin from the EHS tumor and fusing the rat spleen cells with mouse NS-1 myeloma cells. Laminin fragments were generated by proteolytic digestion with thrombin, thermolysin, and chymotrypsin. Monoclonal antibody binding fragments were identified by immunoblotting. Fragments which bound monoclonal antibody LMN-1 included a 440-kilodalton (kDa) chymotrypsin fragment and thermolysin fragments of 440 and 110 kDa. These fragments could also be generated from within a 600-kDa thrombin fragment. Digestion of the 440-kDa chymotrypsin fragment with thermolysin generated the 110-kDa antibody binding fragment and a 330-kDa nonbinding fragment. Immunoblotting was performed on extracts of PYS-2 cells and EHS cells using polyclonal and monoclonal antibodies to laminin. Polyclonal antibodies stained the intact 850-kDa complex and the 200- and 400-kDa subunits, while monoclonal LMN-1 stained only the 400-kDa subunit and the complete molecule. Rotary shadowing of monoclonal LMN-1 bound to laminin molecules indicated that the binding site was within the long arm of laminin. Changes in the model of the internal organization of the laminin molecule are proposed, based on the binding of LMN-1 to the 400-kDa subunit and specific proteolytic fragments. The locations of the major thrombin and chymotrypsin fragments in the model are rotated 180 degrees relative to the previously described model [Ott, U., Odermatt, E., Engel, J., Furthmayr, H., & Timpl, R. (1982) Eur. J. Biochem. 123, 63-72] to include part of the 400-kDa subunit of laminin.     

Novel Angiotensin I-converting enzyme inhibitory peptides found in a thermolysin-treated elastin with antihypertensive activity

Angiotensin I-converting enzyme (ACE) inhibitory activity was generated from elastin and collagen by hydrolyzing with thermolysin. The IC50 value of 531.6 μg/mL for ACE inhibition by the elastin hydrolysate was five times less than 2885.1 μg/mL by the collagen hydrolysate. We confirmed the antihypertensive activity of the elastin hydrolysate in vivo by feeding spontaneously hypertensive rats (male) on a diet containing 1% of the elastin hydrolysate for 9 weeks. About 4 week later, the systolic blood pressure of the rats in the elastin hydrolysate group had become significantly lower than that of the control group. We identified novel ACE inhibitory peptides, VGHyp, VVPG and VYPGG, in the elastin hydrolysate by using a protein sequencer and quadrupole linear ion trap (QIT)-LC/MS/MS. VYPGG had the highest IC50 value of 244 μM against ACE and may have potential use as a functional food.     

Inhibition of thermolysin by 3-trimethylsilylalanine derivatives

The inhibition of thermolysin by an optically active silicon-containing amino acid, 3-trimethylsilylalanine (TMS-Ala), and its derivatives was examined by considering the similarity of structure between TMS-Ala and leucine. Although free l- and d-TMS-Ala did not show the inhibition, several derivatives of l-TMS-Ala, especially Z-l-TMS-Ala and l-Leu-(l-TMS-Ala), exhibited a higher inhibitory activity toward thermolysin than did Z-l-Leu and l-Leu-l-Leu respectively. Effects of TMS-Ala on the activity of its derivatives and the mode of interaction between the derivatives of TMS-Ala and thermolysin are also discussed. Received: 24 February 1999 / Received last revision: 4 June 1999 / Accepted: 27 June 1999     

Limited proteolysis of thermolysin by subtilisin: isolation and characterization of a partially active enzyme derivative

Incubation of the neutral metalloendopeptidase thermolysin at pH 9-10 in the presence of 10 mM CaCl2 for 2 days at room temperature with subtilisin at a 50:1 molar ratio leads to a derivative possessing lower (approximately 3%) but intrinsic catalytic activity. This derivative, called thermolysin S, was isolated by gel filtration in approximately 80% yield and then separated from some residual intact thermolysin by an affinity chromatographic step on Sepharose-Gly-D-Phe. It was found that thermolysin S results from a tight association of two polypeptide fragments of apparent Mr of 24000 and 10000. Dissociation of the complex was achieved under strong denaturing conditions, such as gel filtration on a column equilibrated and eluted with 5 M guanidine hydrochloride. The positions of the clip sites were defined by amino acid analysis, end-group determination, and amino acid sequencing of the isolated fragments and shown to lie between Thr-4 and Ser-5, between Thr-224 and Gln-225, and also between Gln-225 and Asp-226. Thermolysin S, which is therefore a stable complex of fragments 5-224(225) and 225(226)-316, shows a shift in optimum pH of about 1 unit toward the acid range with respect to intact thermolysin and a Km essentially unchanged, with furylacryloyl-Gly-Leu-NH2 as substrate. Inhibitors of thermolysin such as ethoxyformic anhydride and Zn2+ ions inactivate also the nicked enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of dipeptidyl carboxypeptidase and endopeptidase activities in the three enkephalin-hydrolysing metallopeptidases: "angiotensin-converting enzyme", thermolysin and "enkephalinase"

Angiotensin-converting enzyme (ACE), thermolysin and "enkephalinase", three metallopeptidases cleaving the Gly3-Phe4 amide bond of enkephalins, were compared regarding substrate specificity and effects of butanedione, an arginyl-directed reagent. The hydrolysis of enkephalins and analogues was more affected by the nature of P1 and P2 residues in the case of thermolysin than in those of ACE or "enkephalinase"; amidation of the C-terminal carboxylate decreased drastically the hydrolysis by ACE but only marginally by thermolysin and the effect was intermediate for "enkephalinase". With adequate model substrates, the ratio of dipeptidylcarboxypeptidase to tripeptidylcaroxypeptidase (endopeptidase) activities were of 25 for ACE, 3 for "enkephalinase" and only 0.3 for thermolysin. Finally a butanedione treatment increased thermolysin activity, but abolished ACE activity; it reduced "enkephalinase" activity by 80% when measured with a free C-terminal carboxylate enkephalin analogue but only slightly with the corresponding amidated derivative. A critical role of an Arg residue in ACE and, to a lesser extent, in "enkephalinase" (but not in thermolysin) is suggested to be responsible for the preferential dipeptidylcarboxypeptidase activity of these two enzymes.     

Insect inhibitors of metalloproteinases

Two types of peptidic metalloproteinase inhibitors have recently been identified in insects. A homologue of vertebrate tissue inhibitors of metalloproteinases (TIMPs) was found in the fruitfly Drosophila melanogaster which may contributes to regulation of a corresponding matrix metalloproteinase (MMP). The first member of MMPs from insects which shares similarity with vertebrate MMPs has also been cloned and characterized from Drosophila, suggesting conserved evolution of both MMPs and TIMPs. The first insect inhibitor of metalloproteinases (IMPI), which was identified in larvae of the greater wax moth, Galleria mellonella, shares no sequence similarity with known vertebrate or invertebrate proteins and represents the first non-TIMP-like inhibitor of metalloproteinases reported to date. In contrast to TIMPs, the IMPI is not active against MMPs but inhibits microbial metalloproteinases such as bacterial thermolysin. Insects may recognize such toxic metalloproteinases associated with invading pathogens by particular peptidic fragments that result from their nonregulated activity within the hemolymph. Metalloproteinases induce expression of the IMPI along with other antimicrobial proteins in course of humoral immune response of G. mellonella, thereby mediating regulation of metalloproteinase activity released within the hemolymph and inhibition of pathogen development as well.     

Novel Angiotensin I-Converting Enzyme Inhibitory Peptides Found in a Thermolysin-Treated Elastin with Antihypertensive Activity

Angiotensin I-converting enzyme (ACE) inhibitory activity was generated from elastin and collagen by hydrolyzing with thermolysin. The IC₅₀ value of 531.6 µg/mL for ACE inhibition by the elastin hydrolysate was five times less than 2885.1 µg/mL by the collagen hydrolysate. We confirmed the antihypertensive activity of the elastin hydrolysate in vivo by feeding spontaneously hypertensive rats (male) on a diet containing 1% of the elastin hydrolysate for 9 weeks. About 4 week later, the systolic blood pressure of the rats in the elastin hydrolysate group had become significantly lower than that of the control group. We identified novel ACE inhibitory peptides, VGHyp, VVPG and VYPGG, in the elastin hydrolysate by using a protein sequencer and quadrupole linear ion trap (QIT)-LC/MS/MS. VYPGG had the highest IC₅₀ value of 244 µM against ACE and may have potential use as a functional food.     

The effect of proteolytic removal of the C-terminal fragment of rhodopsin on its ability to activate visual cascade

The role of the C-terminal domain of rhodopsin in the activation of transducin was studied. The treatment of photoreceptor membranes with trypsin, thermolysin, and Asp-N-endoprotease led to the respective rhodopsin species devoid of 9, 12-, or 19-aa C-terminal fragments. It was shown that the removal of 9-aa fragment by trypsin does not affect the catalytic activity of the receptor, whereas the thermolysin-induced truncation of the rhodopsin C-terminus by 12 aa about 1.5-fold enhances its activity. The Asp-N-endoprotease-assisted removal of 19 aa (i.e., the shortening by seven more C-terminal aa) virtually unchanges the rhodopsin catalytic activity compared to the preparation truncated with thermolysin. These results suggest that the part of the rhodopsin C-terminal fragment between the sites of its cleavage by trypsin and thermolysin (Val337-Ser338-Lys339) inhibits the signal transduction from rhodopsin to the next component of visual cascade. The English version of the paper.     

Proteolysis of the 85-kilodalton crystalline cysteine proteinase inhibitor from potato releases functional cystatin domains.

The protein crystals found in potato (Solanum tuberosum L.) tuber cells consist of a single 85-kD polypeptide. This polypeptide is an inhibitor of papain and other cysteine proteinases and is capable of binding several proteinase molecules simultaneously (P. Rodis, J.E. Hoff [1984] Plant Physiol 74: 907-911). We have characterized this unusual inhibitor in more detail. Titrations of papain activity with the potato papain inhibitor showed that there are eight papain binding sites per inhibitor molecule. The inhibition constant (Ki) value for papain inhibition was 0.1 nM. Treatment of the inhibitor with trypsin resulted in fragmentation of the 85-kD polypeptide into a 32-kD polypeptide and five 10-kD polypeptides. The 32-kD and 10-kD fragments all retained the ability to potently inhibit papain (Ki values against papain were 0.5 and 0.7 nM, respectively) and the molar stoichiometries of papain binding were 2 to 3:1 and 1:1, respectively. Other nonspecific proteinases such as chymotrypsin, subtilisin Carlsberg, thermolysin, and proteinase K also cleaved the 85-kD inhibitor polypeptide into functional 22-kD and several 10-kD fragments. The fragments obtained by digestion of the potato papain inhibitor with trypsin were purified by reverse-phase high-performance liquid chromatography, and the N-terminal amino acid sequence was obtained for each fragment. Comparison of these sequences showed that the fragments shared a high degree of homology but were not identical. The sequences were homologous to the N termini of members of the cystatin superfamily of cysteine proteinase inhibitors. Therefore, the inhibitor appears to comprise eight tandem cystatin domains linked by preteolytically sensitive junctions. We have called the inhibitor potato multicystatin (PMC). By immunoblot analysis and measurement of papain inhibitory activity, PMC was found at high levels in potato leaves (up to 0.6 microgram/g fresh weight tissue), where it accumulated under conditions that induce the accumulation of other proteinase inhibitors linked to plant defense. PMC may have a similar defensive role, for example in protecting the plant from phytophagous insects that utilize cysteine proteinases for dietary protein digestion.     

Structural basis of the endoproteinase-protein inhibitor interaction

Proteolytic enzymes are potentially hazardous to their protein environment, so that their activity must be carefully controlled. Living organisms use protein inhibitors as a major tool to regulate the proteolytic activity of proteinases. Most of the inhibitors for which 3D structures are available are directed towards serine proteinases, interacting with the active sites in a 'canonical' i.e. substrate-like manner via an exposed reactive site loop of conserved conformation. More recently, some non-canonically binding serine proteinase inhibitors directed against coagulation factors, in particular thrombin, a few cysteine proteinase inhibitors inhibitory towards papain-like proteinases, and three zinc endopeptidase inhibitors directed against metzincins and thermolysin have been characterised in the free and complexed state, displaying novel mechanisms of inhibition with their target proteinases. These different interaction modes are presented and briefly discussed with respect to the different strategies applied by nature.     

Molecular mechanism of the inhibitory effect of cobalt ion on thermolysin activity and the suppressive effect of calcium ion on the cobalt ion-dependent inactivation of thermolysin

Thermolysin activity in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide (FAGLA) and FA-l-leucyl-l-alanine amide (FALAA) was examined at various Co(2+) and Ca(2+) concentrations. It decreased to 28% with increasing [Co(2+)] up to 18 mM. The Co(2+)-dependent inactivation was in part suppressed by adding Ca(2+) ion up to 0.5 mM, but 33% of the activity remained to be inactivated even with a sufficient concentration of Ca(2+) (>0.5 mM). The Co(2+)-dependent inactivation was shown to be composed of Ca(2+)-sensitive and Ca(2+)-insensitive parts. In the latter part which is observed at [Ca(2+)] >0.5 mM, Co(2+) plays as a competitive inhibitor. On the other hand, the Co(2+)-dependent inactivation in the Ca(2+)-sensitive part observed at [Ca(2+)] <0.5 mM proceeds time-dependently following second-order kinetics, and the time-course is in good agreement with that of decrease in the thermolysin band due to autolysis in SDS-PAGE. This indicates that Co(2+) accelerates the autolysis. Here, we describe the co-regulation of thermolysin activity by Co(2+) and Ca(2+) ions and propose a molecular mechanism for the inhibition of thermolysin by Co(2+) and suppressive effect of Ca(2+) on the Co(2+)-dependent inhibition. Co(2+) ion inhibits thermolysin activity not only as a competitive inhibitor but also promoting the autolysis.     

Characterization of a 59 kDa gelatin-binding fragment of buffalo plasma fibronectin

Limited proteolysis of buffalo plasma fibronectin (FN) by thermolysin yielded four gelatin-binding fragments of which, the major 59 kDa fragment, GBF1, was isolated by gelatin-Sepharose and heparin-Sepharose affinity columns. GBF1 appeared during early phase of thermolysin digestion and remained intact even after 4 hr of digestion. GBF1 may be similar to 56 kDa gelatin-binding fragment of FNs from human and hamster plasma. But, it is more resistant to thermolysin cleavage. The fragment binds to heparin with low affinity. On the basis of the structure of human plasma FN, the modular structure of GBF1 may be given as: 6Fn1 1Fn2 2Fn2 7Fn1 8Fn1 9Fn1 1Fn3. Biophysical properties of GBF1 suggest an expanded native conformation. The interaction of the fragment with gelatin is pH-dependent and independent of NaCl concentration.     

Effect of divalent metal ions on the digestibility of concanavalin A by endopeptidases.

Demetallized concanavalin A is degraded rapidly at pH 7.0 and 8.2 by alpha-chymotrypsin, thermolysin or trypsin, yielding peptide fragments devoid of ability to bind to Sephadex G-75. Addition of Ni2+ and of Ca2+ confers on concanavalin A high resistance towards proteolytic attack so that even after long periods of exposure to the enzymes, almost all of the saccharide-binding capacity is preserved. Ni2+ alone protects strongly at pH 7.0 but not at pH 8.2. Apparently, both the transition metal ion and Ca2+ play an important role in stabilizing the native conformation of the protein molecule. Digestion of demetallized concanavalin A with alpha-chymotrypsin or thermolysin readily yields small peptide fragments (Mr less than 10 000), while trypsin yields as the major product(s) larger peptide(s) (Mr approximately 20 000) of appreciable resistance to further fragmentation.