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Turnip isoperoxidase TP 7 had its hemin group removed and was cleaved by trypsin. The digest was fractionated by gel filtration and high-voltage paper electrophoresis and yielded 24 peptides, which counted for all 296 amino acid residues of the enzyme. Sequence analyses on the tryptic peptides and, when necessary, on their thermolytic derivatives were carried out by manual Edman degradation followed by dansylation or by quantitative amino acid analysis of thiazolinones convened by HI. The four disulfide bridges and the only site of carbohydrate attachment of turnip peroxidase 7 are located. The present analysis of tryptic peptides has been complemented by cyanogen bromide fragments cleaved by chymotrypsin as described in the accompanying paper. [ABSTRACT FROM AUTHOR]

In a previous paper we have characterized five plant peroxidases, P1, P2, P3 and P7 of turnip and horseradish isoperoxidase C by peptide mapping studies, and only found two highly homologous sequences present in all. Both contained histidine. The findings supported previous suggestions of two histidine sequences near the peroxidase heme prostetic group, in the present paper we present the amino acid sequences around the histidine residues of all four turnip peroxidases, i. e. of 25 residues around the histidine proximal to heine, and 34 residues around the probably distally located histidine, and compare them with the histidine-containing sequences of the complete amino acid sequence of horseradish isoperoxidase C. Substitutions of residues are rare close to these histidines, but more abundant with greater distances. The probably distal sequences of P1, P2, P3 and horseradish peroxidase C all contain two histidine residues, at positions 40 and 42. In P7, however, residue 40 is phenylalanine, a substitution presumably important to its abnormal physico-chemical and enzymic properties. Gel filtration profiles of tryptic digests of the turnip isoperoxidases confirm their previous classification into a P1, P2 and P3 group and a distinct P7 enzyme, but further prove the presence of several sites of carbohydrate attachment in P1, P2 and P3 peroxidases, like in horeseradish peroxidase C which has eight sites. P7 has one such site. [ABSTRACT FROM AUTHOR]

In paper [5] a simplified model for the hydrophobic binding sites of trypsin was proposed, showing where 1-amino-n-alkanes and α,ω-diamino-n-alkanes are bound while they competitively inhibit the catalyzed hydrolysis of a substrate ester. In the model, the enzyme bas two hydrophobic binding sites separated by an aqueous region which presumably contains the catalytic important residues. The first site binds up to 4 methylene groups of the aliphatic side chain adjacent to the ω-amino group: additional—CH2 groups in t, he chain extend into the aqueous region. If the chain is larger than 7 carbon atoms, the additional methylene or methyl groups are bound to the second hydrophobic site. This site is found to bind less strongly than the first site. This paper presents a thermodynamic investigation of the binding properties of trypsin in the formation of complexes with cationic esters and amids which bear an ammonium group on either the end of the acid side chain or the end of the alcohol or amine component. Free energy and in addition enthalpy and entropy of binding were measured as a function of the length of the inhibitor molecule and as a function of the position of the ester or amide carbonyl group. It was found that thc stability of a complex increases linearly with length of the molecule, and that the magnitude of increase depends on the position of the carbonyl group. Amides always exhibit a smaller increase than esters. Inhibitors with the ammonium group on the alcohol component, were bound with about, the same stability than the corresponding ω-aminoacidesters. The results are consistent, with the model. Positioning of the carbonyl group within the first or second binding site reduces stability due to restricted interaction of the hydrophobic residues on enzyme and inhibitor. Only when the carbonyl group falls into the aqueous region, the free energy for an inhibitor containing the ester group is similar to that for a 1-amino-n-alkane with identical chain length. Enthalpy and entropy as functions of the position of the carbonyl group show minima for the position within the aqueous region. This observation is correlated with the jump of enthalpy and entropy of complex formation of trypsin in the series of 1-amino-n-alkanes reported earlier. It is shown that the magnitude of free energy of complex formation with specific substrates is well estimated on basis of the model. As a consequence, the alternative binding of either the 2-N-acyl group or the alcohol or amine component, to the second hydrophobic site may imply a mode of less productive binding. It is emphasized that unproductive binding should be reflected in the Michaelis Menten constants for substrate amides whereas thc Michaelis Menten constant, for a substrate ester is normally not perturbed. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the heme-binding domains of rat liver cytochromes b5 from outer mitochondrial membranes and from microsomes has been determined by a combination of automatic and manual degradation of fragments generated by trypsin digestion and by cleavage at tryptophan. Tryptic peptides were separated by high-pressure liquid chromatography. The sequence of microsomal cytochrome b5 is identical with the one published by Owls and Heinemann after completion of this study [Biochim. Biophys. Acta (1982) 704. 163- 173]. The sequence of outer membrane cytochrome b5 differs from the microsomal one at 38 positions out of 91. There are 40 positions invariant between this sequence and the eight micrasornal sequences published thus far. The non-conservative substitutions are located at the surface of the known three-dimensional structure of calf microsomal cytochrome b5 except for the substitution of histidine- 15 by arginine. This paper brings the final proof that two iso-cytochromes b5- exist in the same cell. Their high degree of similarity as well as their differential cellular localization raise some questions which are briefly discussed. [ABSTRACT FROM AUTHOR]

Our previous studies [1,2] on maleyl bovine αS1-B casein were concerned with the isolation and amino acid composition of the tryptic peptides designated as Tm peptides. The sequence of the COOH-terminal Tm peptide containing 48 residues was also reported. In the present study, we report the overlaps of all the cyanogen bromide peptides as well as the Tm peptides from αs1-B casein. The αs1-B casein which contains five methionyl residues was cleaved with cyanogen bromide. The six expected peptides obtained by this treatment and designated as CN peptides, were separated initially on Dowex-50X2 and further purified by Sephadex column chromatography. The amino acid composition of these six purified CN peptides was determined. The results were in perfect agreement with the amino acid composition of αs1-B casein deduced from the analysis of Tm peptides. The three Tm peptides from αs1-B casek@ which contained methionyl residues were also cleaved by cyanogen bromide. The fragments obtained were purified by either paper chromatography or Sephadex column chromatography and the amino acid composition of these purified peptides was determined. Of the eight peptides thus obtained, three were found identical with the CN peptides obtained directly by BrCN treatment of the αs1-B ease@. The remaining three CN peptides from αs1-B casein for which identical fragments could not be located fun the Tm peptides, were digested with trypsin (in two eases after maleylation) and the resulting fragments were purified by Sephadex column chromatography. These trifle peptides provided the remaining gaps. This permitted in to locate the relative position of all the CN peptides in the αs1-B casein molecule. These results also indicate the location of all the Tm peptides except 2 in the αs1-B casein molecule. Further, the chymotryptic peptides from one of the CN fragments of αs1-B casein provided the missing overlaps. [ABSTRACT FROM AUTHOR]

In order to determine the primary structure of the bovine β-caseins, the A2 variant was first cleaved with trypsin and cyanogen bromide (CNBr). The tryptic hydrolyzate was separated by column chromatography on Dowex-50. After additional purifications, 13 peptides numbered Ti to T13 were isolated. The 14th tryptic peptide, T14, not eluted from the column, was directly obtained from the hydrolyzate by preparative paper electrophoresis. Amino-acid analyses were carried out on all these peptides. Peptide T1 contain 2 Arginyl residues, one of which is NH2-terminal. This peptide represents the NH2-terminal end of the β-casein. Its composition is identical to that reposed by Peterson et al. in 1958 [1] for a phosphopeptide isolated from the β-casein. Peptide T11 contains 2 Lysyl residues, one of which is NH2terminal. The COOH-terminal peptide was identified with peptide T4, devoid of basic aminoacids. The 14 tryptic peptides account for all the residues of the β-casein A2 when compared with the already published amino-acid analyses of the whole protein [2, 3]. Several other peptides, originating from incomplete or non-specific cleavages, were also obtained. Similarly, 7 peptides accounting for the 6 methionyl residues of the protein were obtained from the same variant of β-casein after cleavage with cyanogen bromide. Six of these peptides were separated on Sephadex G-50. The seventh peptide was obtained by chromatography on Dowex-50 of the whole CNBr-digest. After additional purifications, the ammo-acid compositions of the 7 pure peptides were determined. Peptide CN1 contains peptide T1. It represents the NH2terminal end of β-casein. The COOH-terminal end of β-casein was identified with peptide CN7. As in the case of the tryptic peptides, the 7 CNBr-peptides account for all of the amino-acid residues of β-casein A2. β-casein A2 has 208 amino-acid residues. Its molecular weight is very close to 24 000. [ABSTRACT FROM AUTHOR]

Cholecystokinin­pancreozymin has been found to have the partial structure Lys (Ala1, Gly1, Pro1, Ser1) Arg Val (Ile1, Met1, Ser1) Lys Asn (Asx1, Glx1, His1, Leu2, Pro1, Ser2) Arg. Ile (Asp1, Ser1) Arg Asp [Gly1, Met2, Trp1, Tyr (SO3H)] Asp Phe NH2. Of the four peptide bonds in this polypeptide, three arginyl and one lysyl, that are cleaved by trypsin only the arginyl-valine bond is cleaved by thrombin, with retention of the cholecystokinin and pancreozymin activities of the original molecule in the C-terminal heptacosapeptide. The C-terminal tryptic octapeptide too has strong cholecystokinin and pancreozy-min activity. Except for the replacement of a threonine with methionine the eight amino acid units of this peptide are the same as those that comprise the corresponding part of the molecule of caerulein. Also like that in caerulein, the phenolic group of the only tyrosine of cholecystokinin-pancreozymin is esterified with sulphuric acid. [ABSTRACT FROM AUTHOR]

Sequencing of chyinotrypsin, trypsin, collagenase- and hydroxylamine-derived peptides, using the automated Edman degradation procedure, yielded the complete amino acid sequence of α2-CB4 from calf skin collagen (321 residues). Together with the data from earlier work, an uninterrupted sequence in the helical region of the α2-chain from residues 1-393 is now known. Glycine is found in every third position of the peptide. Hydroxylation of proline and lysine occurs only in the Y-position of the triplet Gly-X-Y and is not complete in every position. Some residues, such as glutamic acid, leucine, phenylalanine and arginine, are distributed non-randomly between the X and Y-positions and this non-random distribution is different in the α1 and α2-chains. Comparison of the N-terminal 393 residues from the helical region of the α1 and α2-chains revealed a nearly identical distribution of charged polar residues arginine, lysine, glutamic and aspartic acids. The distribution of the triplet Gly-Pro-Hyp is similar in both chains. The remaining residues in the α2-chain exhibit a high degree of substitutions when compared with those in the α1-chain. Approximately one in every two residues in both the X and Y-positions are substituted. [ABSTRACT FROM AUTHOR]

For the elucidation of the amino-acid sequence of the carboxyl-terminal region of soybean trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel filtration on Bio-Gel P-4. Further fractionation and purification of the peptides were performed by ion-exchange chromatography on Dowex 1X2, by gel filtration on Bio-Gel P-2 or by high-voltage paper electrophoresis at pH 1.9 and 3.6. Three peptides were obtained in pure form from fragment C and ten peptides from fragment D, and their amino-acid sequences were determined by the direct Edman method and by carboxy- peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptic peptides from fragment D were obtained from a chymotryptic hydrolysate of fragment D. Nine main peptides and rune minor peptides were obtained. The amino acid composition and the partial amino-acid sequence of the 18 chymotryptic peptides of fragment D made it possible to establish the amino-acid sequence of the carboxyl-terminal region of the inhibitor. The complete amino-acid sequence of soybean trypsin inhibitor (Kunitz) deduced here was compared with that of Bowman-Birk inhibitor, another well-known soybean proteinase inhibitor. [ABSTRACT FROM AUTHOR]

For the elucidation of amino acid sequence around the reactive site of soybean trypsin inhibitor (Kunitz), fragments A and B were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel filtration on Bio-Gel P-4. Further purification of the peptides was carried out by gel filtration on Sephadex G-25 or by high-voltage paper electrophoresis at pH 3.6 and 6.5. Nine peptides were obtained in pure form from fragment A and three peptides from fragment B, and their amino acid sequences were determined by the direct Edman method and by carboxy-peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptie peptides from fragments A and B were obtained from a chymotryptic hydrolysate of fragment AB by gel filtration on Bio-Gel P-4, and their ammo acid compositions and sequence analyses of some peptides made it possible to establish the amino acid sequence of an amino-terminal region of the inhibitor consisting of AB amino acid residues. The reactive site (Arg63-Ile64) of the inhibitor to trypsin was involved in this region. [ABSTRACT FROM AUTHOR]

The C-terminal fragment of hog pepsin, which had been obtained in the preceding study on the cyanogen bromide hydrolysate of this protein, was subjected to sequential studies. The 37-residue peptide was digested in independent experiments by trypsin, chymotrypsin, and pepsin, and the amino acid sequence of the arising peptides was determined. In parallel experiments the sequence of seventeen amino acid residues in the N-terminal region of the peptide was established by the Edman degradation technique. The obtained data permit the C-terminal peptide of hog pepsin to be ascribed the amino acid sequence Asp-Val-Pro-Thr-Ser-Ser-Gly-GluLeu-Trp-Ile-Leu-Gly-Asp-Val-Phe-Ile-Arg-Gln-Tyr-Tyr-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-LysVal-Gly-Leu-Ala-Pro-Val-Ala. These results extend and pauly correct our previous knowledge of this region of the pepsin molecule as recorded in literature. The problem of the tryptophan content of pepsin is briefly discussed. [ABSTRACT FROM AUTHOR]

After pronase treatment of the murein-lipoprotein complex (rigid layer) of the cell wall of E, coli B or E. coli K12 (W 945), lysine and arginine remain as the sole amino acids covalently bound to the murein (peptidoglycan, glycopeptide). These amino acids occur in equimolar amounts, each equal to the amount of lysine remaining with the murein after trypsin digestion of the murein-lipoprotein complex. From partial acid hydrolysates of such a murein, prepared by pronase digestion of the murein-lipoprotein complex, the following peptides have been isolated: (1) diaminopimelyl-lysyl-arginine; (2) alanyl-glutamyl-diaminopimelyl-lysyl-arginine; (3) glucosaminyl-muramyl-alanyl-glutamyl-diaminopimelyl-lysyl-arginine. Peptide 1 shows that the lipoprotein is bound by the α-amino group of the presumably N-terminal lysine to the carboxyl group of diaminopimelic acid. Peptide 2 consists of a peptide side chain of the murein to which the two amino acids of the N-terminal end of the lipoprotein, lysine and arginine, are attached. Peptide 3 constitutes a repeating unit of the murein to which the peptide lysyl-arginine of the lipoprotein is bound. The lipoprotein cleaved from the murein by a short trypsin digestion had an amino acid composition similar to the lipoprotein in the untreated murein-lipoprotein complex and arginine as N-terminal amino acid. The following structure is proposed: murein-lysyl-arginyl-lipoprotein. In the rapid reaction of trypsin with the cell wall the enzyme apparently cleaves at the C-terminal end of the lysine of the murein-lipoprotein linkage which results in murein-lysine and arginyl-lipoprotein. Some cleavage at the C-terminal end of arginine gives rise to free arginine (14%). These results support the supramolecular structure of the murein-lipoprotein complex previously proposed. On the average one lipoprotein molecule is covalently bound to every tenth repeating unit of the murein from which an average distance of 103 Å between two lipoprotein molecules along the polysaccharide chains of the murein is deduced. [ABSTRACT FROM AUTHOR]

A protein with pancreastatin-tike immunoreactivity has been isolated and purified from liver metastasis of a patient with insulinoma. NH2-terminal residue analysis, in conjunction with the use of antibodies that are specific for the C-terminal amide peptide of porcine pancreastatin, identified this protein as a 186-amino-acid protein corresponding to human chromogranin A-1 16-301 (the fragment corresponding to the positions from 116 to 301 of human chromogranin A). Digestion of this protein with trypsin yielded a 48-amino-acid peptide with the retention of full pancreastatin activity. Serum from patient with insulinoma contains a peptide specie(s) that comigrates with the 48-amino-acid pancreastatin, suggesting that this peptide might be a physiologically important circulation form of pancreastatin in humans. A sensitive radioimmunoassay was established using antibody developed against a synthetic 29-amino-acid peptide amide of pancreastatin. Immunocytochemical staining revealed that a major population of human pancreatic islet cells were immunoreactive to the antiserum but with varying intensity of staining. Pancreastatinlike immunoreactivity was not observed in exocrine acinar cells. [ABSTRACT FROM AUTHOR]

Acanthamoeba profilin-II [Kaiser, D. A., Sato, M., Ebert, R. F. and Pollard, T. D. (1986) J. Cell. Biol. 102, 221-226] was digested with trypsin or cleaved by 2-(2-nitrophenylsulphenyl)-3-methyl-3-bromoindolenine. The tryptic peptides were purified by reversed-phase-high-performance liquid chromatography and completely sequenced using automated gas-phase sequence analysis. The complete profilin-II sequence was deduced by ordering the tryptic peptides using the sequence information of the tryptophan-cleavage products. Acanthamoeba profilin II was found to be homologous to the previously determined profilin-I sequence [Ampe, C., Vandekerckhove, J., Brenner, L., Tobacman, L. and Korn, E. D. (1985) J. Biol. Chem. 260, 834-840]. Like profilin-I, profilin-II consists of 125 amino acids, has a blocked NH2 terminus and a trimethyllysine residue at position 103. ProfilinII differs in at least 21 positions from one of the profilin-I isoforms. The amino acid exchanges are mainly concentrated in the middle part of the sequence. Profilin-II contains two more basic residues than profilin-I, which explains its higher isoelectric point. [ABSTRACT FROM AUTHOR]

Two-dimensional f-resolved 1H NMR spectroscopy was used to measure the vicinal spill-spin coupling constants 3JHαHβ for numerous, previously individually assigned amino acid residues in the basic pancreatic trypsin inhibitor at various temperatures between 30 and 85°C. An analysis of this data is proposed which enables one to compare the spatial arrangements of individual amino acid side chains in solution and in single crystals of the protein, and which also provides information on the mobility of the side chains in the solution conformation. As a rule, the amino acid side chains in the interior of the protein were found to be locked into unique spatial orientations, with the mobility restricted to rapid rotational fluctuations about this unique value for the dihedral angle χ1. In most, but not all, instances the data for the interior amino acids indicate identical average conformations tor the amino acid side chains in single crystals and in solution. For residues on the protein surface structural rearrangements between crystal and solution appear to be common, and the mobility in the solution conformation may include rapid averaging between two or several distinct, preferentially populated values of χ1 analogous to the gauche-trans-gauche isomerization in isolated amino acids [ABSTRACT FROM AUTHOR]

Two trypsin inhibitors from the seed or Acacia elata, a legume of the subfamily Mimosoideae, were isolated by affinity chromatography on trypsin-Sepharose 48 and separated by chromatography on SP-Sephadex C-25 and Sephadex G-100. The two inhibitors, with molecular weights of about 20000, were composed of two polypeptide chains linked by a disulfide bond. The two inhibitors had essentially the same amino acid compositions and both contained four half-cystine residues, no methionine and were rich in aspartic acid, glutamic acid, glycine and leucine. The inhibitors had isoelectric points of 6.4 and 5.9. The inhibitors stoichiometrically inhibited trypsin in the molar ratio of 1: 1, α-chymotrypsin was inhibited also in a 1: 1 molar ratio but the binding of the enzyme by the inhibitor was weaker and the inhibitor-chymotrypsin complex dissociated during the assay. Both enzymes are probably inhibited at an identical site. Amino-terminal sequence analysis of the two polypeptide chains of the inhibitors revealed extensive homology with the trypsin inhibitor from the silk tree (another Mimosoideae legume); both these inhibitors are homologous with the soybean trypsin inhibitor (Kunitz). [ABSTRACT FROM AUTHOR]

The kinetic parameters of the tryptic hydrolysis of tripeptidyl-p-nitroanilide substrates were determined and the data were studied by regression analysis. The sequence of substrates optimal from the viewpoint of kinetic constants 1/Km, kcat and kcat/Km was established and the influence of amino acid side chains on the binding and reactivity of substrates was calculated. At subsite P3 At subsite P3[notation of Schechter and Berger (1967) Biochem, Biophys, Res. Commun. 27, 157] polar side chains (Asn, D-Arg) are favourable as regards 1/Km, whereas hydrophobic side chains are preferred definitely from the viewpoint of catalytic efficiency, just as at subsite P2. In the side chain contributions, calculated for the kinetic parameters, the P3-S3 interaction predominates, in spite of the fact that the properties of the residue at subsite Pi decide whether hydrolysis occurs at all. The ZAsn-Ile-Arg-Nan sequence was predicted as a better substrate than those tested experimentally. The compound was synthesized, and the calculated value of its 1/Km (116.4 mM-1) was in a good agreement with the measured value (100.2 mM-1). Comparing the data obtained with trypsin with those observed with thrombin, elastase and substrates, we can establish that the homology of these enzymes can be characterized at each binding subsite by the aid of tripeptidyl- p-nitroanilide substrates. The quantities derived allow one to envisage a novel type of comparison of the proteases. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of the u chain of the mitogenic lectin from Vicia sativa has been determined. The polypeptide was digested by trypsin and chymotrypsin. The fragments were isolated and most of them were sequenced by automatic solid-phase Edman degradation. A total sequence of 52 amino acid residues was obtained which is homologous to the α subunits of the lectins from Pisum sativum, Lens culinaris and Vicia faba. The central part (residues 13–32) in particular is completely conserved in all four proteins. In contrast to the three other known sequences, the V. sativa α subunit has an additional serine at the N terminus. The differences of the related amino acid sequences of these lectins and concanavalin A, the lectin from Canavalia ensiformis, have been compared using the relative substitution frequency found in homologous proteins by McLachlan. The degree of homology decreases in the order: V. sativa > P. sativum > V. faba > L. culinaris > C. ensiformis. Prediction of the secondary structure according to Chou and Fasman reveals that the lectin α chain is similar to concanavalin A in the first half of tile molecule whereas the C-terminal part apparently tends to form an α helix. [ABSTRACT FROM AUTHOR]

Binding of tRNAf, Met to the monomeric trypsin-modified methionyl-tRNA synthetase turns off the methionine-dependent isotopic ATP-PPi exchange. In the case of the dimeric native methionyl-tRNA synthetase, one anticooperatively bound tRNAt, Met inhibits the exchange by only 50%. These behaviours of tRNA do not require the integrity of the 3′-terminal adenosine. Esterification by methionine of the 3′ end of tRNA reinforces the affinity of tRNAf, Met for the enzymes. In the case of the native enzyme, due to this effect, a second binding mode for methionyl-tRNA may be demonstrated through the isotopic exchange. This additional binding of tRNA corresponds to the expression of the anticooperatively blocked tRNA binding site. Methionine reverses competitively the reinforcing effect of the esterified methionyl moiety on tRNA binding. It is concluded that after esterification of tRNA, the aminoacyl residue still binds the enzyme, probably within the methionine activating site. The latter behaviour may account for the observation that excess methionine accelerates the aminoacylation turnover rate of tRNAf, Met. [ABSTRACT FROM AUTHOR]

Specific and non-specific trypsin substrates of known structure have been examined for common features. This analysis suggests that trypsin has a specificity for a particular conformation near the scissile bond which I denote as a conformational specificity. This conformation is a bent left-handed helix at the third and fourth (P3 and P4) amino acid positions toward the amino terminus from the scissile bond which I denote as a conformational specificity. This conformation is a bent left-handed show a high frequency of proline and glycine at these positions consistent with the left-handed helical conformation. This apparent secondary specificity for a particular substrate residue other than that at the primary position is not related to the nature of the residues at the third and fourth positions. Rather, these residues determine the bend of left-handed helix which has the effect of exposing main chain hydrogen-bonding groups of the substrate peptide chain to hydrogen-bonding groups on the enzyme. Thus, the secondary specificity of trypsin is not sequence-specific, but is for peptide main chain in the third and fourth positions and is determined by the tertiary structure of the substrate. This hypothesis for conformational and secondary specificity in trypsin can be extended to chymo- trypsin. It also provides a means for the regulation of certain processes in vivo catalyzed by other proteases. [ABSTRACT FROM AUTHOR]

The six isoinhibitors of trypsin and chymotrypsin which were previously purified from a crude extract of chick peas were further studied with the intention of establishing the relationships among them. Exposure of each fraction to active, matrix-bound trypsin resulted in a decrease of the specific inhibitory activities towards trypsin of fractions P-l, P-3 and P-5 while those of P-4 and P-6 were increased. Each of these fractions was separated into several sub-fractions after chromatography on a DEAE-Sephadex A-25 column under conditions identical to those used previously for their purification. The elution positions, specific inhibitory activities towards trypsin and electrophoretic patterns in polyacrylamide gels of the various sub-fractions were determined. On the basis of the results thus obtained it is concluded that fractions P-3, P-4, P-5 and P-6 originate from a common inhibitor molecule. From the amino acid compositions and amino- terminal sequences of these four fractions, it is further concluded that P-3 and P-4 are identical proteins and that P-5 and P-6 are probably derived from P-3 and P-4, respectively, as a result of tryptic removal of the amino-terminal Val-Lys residues from the latter two fractions. Since the distribution of the products obtained from P-I is different from that obtained from the other four fractions, it is also concluded that P-1 is propably derived from a second inhibitor originally present in chick peas. The inhibitors in chick peas were also isolated by conventional chromatographic procedures. Preliminary results indicate the presence of two inhibitors, both with a molecular weight of around 16000. Since the fractions thus obtained were not homogeneous they were not further characterized. Nevertheless, the findings confirm the conclusions reached above and also show that the inhibitors probably exist as dimers in the seeds of chick peas since each of the isoinhibitors previously isolated had molecular weights of about 10000. [ABSTRACT FROM AUTHOR]