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Item Mapping immunogenic epitopes of an adhesin-like protein from Methanobrevibacter ruminantium M1 and comparison of empirical data with in silico prediction methods.(Springer Nature Limited, 2022-06-21) Khanum S; Carbone V; Gupta SK; Yeung J; Shu D; Wilson T; Parlane NA; Altermann E; Estein SM; Janssen PH; Wedlock DN; Heiser AIn silico prediction of epitopes is a potentially time-saving alternative to experimental epitope identification but is often subject to misidentification of epitopes and may not be useful for proteins from archaeal microorganisms. In this study, we mapped B- and T-cell epitopes of a model antigen from the methanogen Methanobrevibacter ruminantium M1, the Big_1 domain (AdLP-D1, amino acids 19-198) of an adhesin-like protein. A series of 17 overlapping 20-mer peptides was selected to cover the Big_1 domain. Peptide-specific antibodies were produced in mice and measured by ELISA, while an in vitro splenocyte re-stimulation assay determined specific T-cell responses. Overall, five peptides of the 17 peptides were shown to be major immunogenic epitopes of AdLP-D1. These immunogenic regions were examined for their localization in a homology-based model of AdLP-D1. Validated epitopes were found in the outside region of the protein, with loop like secondary structures reflecting their flexibility. The empirical data were compared with epitope predictions made by programmes based on a range of algorithms. In general, the epitopes identified by in silico predictions were not comparable to those determined empirically.Item Structural characterization of a PCP-R didomain from an archaeal nonribosomal peptide synthetase reveals novel interdomain interactions(Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology, 2021-02-17) Deshpande S; Altermann E; Sarojini V; Lott JS; Lee TV; Jez JNonribosomal peptide synthetases (NRPSs) are multimodular enzymes that produce a wide range of bioactive peptides, such as siderophores, toxins, and antibacterial and insecticidal agents. NRPSs are dynamic proteins characterized by extensive interdomain communications as a consequence of their assembly-line mode of synthesis. Hence, crystal structures of multidomain fragments of NRPSs have aided in elucidating crucial interdomain interactions that occur during different steps of the NRPS catalytic cycle. One crucial yet unexplored interaction is that between the reductase (R) domain and the peptide carrier protein (PCP) domain. R domains are members of the short-chain dehydrogenase/reductase family and function as termination domains that catalyze the reductive release of the final peptide product from the terminal PCP domain of the NRPS. Here, we report the crystal structure of an archaeal NRPS PCP-R didomain construct. This is the first NRPS R domain structure to be determined together with the upstream PCP domain and is also the first structure of an archaeal NRPS to be reported. The structure reveals that a novel helix-turn-helix motif, found in NRPS R domains but not in other short-chain dehydrogenase/reductase family members, plays a major role in the interface between the PCP and R domains. The information derived from the described PCP-R interface will aid in gaining further mechanistic insights into the peptide termination reaction catalyzed by the R domain and may have implications in engineering NRPSs to synthesize novel peptide products.Item Self-assembly and hydrogelation of a potential bioactive peptide derived from quinoa proteins(Elsevier BV, 2024-02) Cheng L; De Leon-Rodriguez L; Gilbert EP; Loo T; Petters L; Yang ZIn this work the identification of peptides derived from quinoa proteins which could potentially self-assemble, and form hydrogels was carried out with TANGO, a statistical mechanical based algorithm that predicts β-aggregate propensity of peptides. Peptides with the highest aggregate propensity were subjected to gelling screening experiments from which the most promising bioactive peptide with sequence KIVLDSDDPLFGGF was selected. The self-assembling and hydrogelation properties of the C-terminal amidated peptide (KIVLDSDDPLFGGF-NH2) were studied. The effect of concentration, pH, and temperature on the secondary structure of the peptide were probed by circular dichroism (CD), while its nanostructure was studied by transmission electron microscopy (TEM) and small-angle neutron scattering (SANS). Results revealed the existence of random coil, α-helix, twisted β-sheet, and well-defined β-sheet secondary structures, with a range of nanostructures including elongated fibrils and bundles, whose proportion was dependant on the peptide concentration, pH, or temperature. The self-assembly of the peptide is demonstrated to follow established models of amyloid formation, which describe the unfolded peptide transiting from an α-helix-containing intermediate into β-sheet-rich protofibrils. The self-assembly is promoted at high concentrations, elevated temperatures, and pH values close to the peptide isoelectric point, and presumably mediated by hydrogen bond, hydrophobic and electrostatic interactions, and π-π interactions (from the F residue). At 15 mg/mL and pH 3.5, the peptide self-assembled and formed a self-supporting hydrogel exhibiting viscoelastic behaviour with G' (1 Hz) ~2300 Pa as determined by oscillatory rheology measurements. The study describes a straightforward method to monitor the self-assembly of plant protein derived peptides; further studies are needed to demonstrate the potential application of the formed hydrogels in food and biomedicine.Item Transcriptomic Identification of a Unique Set of Nodule-Specific Cysteine-Rich Peptides Expressed in the Nitrogen-Fixing Root Nodule of Astragalus sinicus(The American Phytopathological Society in cooperation with the International Society for Molecular Plant-Microbe Interactions, 2022-10-08) Wei F; Liu Y; Zhou D; Zhao W; Chen Z; Chen D; Li Y; Zhang X-XLegumes in the inverted repeat-lacking clade (IRLC) each produce a unique set of nodule-specific cysteine-rich (NCR) peptides, which act in concert to determine the terminal differentiation of nitrogen-fixing bacteroid. IRLC legumes differ greatly in their numbers of NCR and sequence diversity. This raises the significant question how bacteroid differentiation is collectively controlled by the specific NCR repertoire of an IRLC legume. Astragalus sinicus is an IRLC legume that forms indeterminate nodules with its microsymbiont Mesorhizobium huakuii 7653R. Here, we performed transcriptome analysis of root and nodule samples at 3, 7, 14, 28 days postinoculation with M. huakuii 7653R and its isogenic ∆bacA mutant. BacA is a broad-specificity peptide transporter required for the host-derived NCRs to target rhizobial cells. A total of 167 NCRs were identified in the RNA transcripts. Comparative sequence and electrochemical analysis revealed that A. sinicus NCRs (AsNCRs) are dominated by a unique cationic group (termed subgroup C), whose mature portion is relatively long (>60 amino acids) and phylogenetically distinct and possessing six highly conserved cysteine residues. Subsequent functional characterization showed that a 7653R variant harboring AsNCR083 (a representative of subgroup C AsNCR) displayed significant growth inhibition in laboratory media and formed ineffective white nodules on A. sinicus with irregular symbiosomes. Finally, bacterial two-hybrid analysis led to the identification of GroEL1 and GroEL3 as the molecular targets of AsNCR067 and AsNCR076. Together, our data contribute to a systematic understanding of the NCR repertoire associated with the A. sinicus and M. huakuii symbiosis.Item Peptide fingerprinting and predictive modelling of fermented milk : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North Campus, New Zealand(Massey University, 2021) Murphy, FionnualaFermented milk products are valued by consumers and the food industry for their nutritional properties, pleasant taste, and texture. Consumer demands and expectations for such products are constantly changing. Understanding how consumers perceive the sensory characteristics of food and the relationship these characteristics have with the chemical components of food can provide insight that can enable food researchers and manufacturers to develop food products that are tailored to provide enhanced sensory qualities. Establishing techniques that allow for in-silico prediction or correlation of sensory qualities can enable a more rapid approach that would aim to enable researchers to meet the demands of consumers. This research firstly explored mass spectrometric techniques for the rapid fingerprinting of milk and fermented milk products, using Matrix-Assisted Laser Desorption Ionisation - Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and Rapid Evaporative Ionisation Mass Spectrometry (REIMS), two technologies that require minimal sample preparation and can rapidly generate a fingerprint of a food’s chemical components. Peptide fingerprints obtained by MALDI-TOF MS and analysed by principal component analysis were effective at discriminating the two fermented milk and milk samples. Supervised discrimination of low molecular weight fingerprints obtained via REIMS and MALDI-TOF MS proved less effective but demonstrated some potential and could be used alongside other analyses in future studies. These techniques were explored with a view to establishing a technique that could provide rapid insights into a food’s chemical composition, and which could also effectively discriminate the chemical components of the product. Such techniques could be used for rapid screening of products and can provide insight into the chemical components that are driving the variation in different products, which may be reflective of the differences in sensory characteristics. Next, peptide fingerprinting and predictive modelling were investigated in milk fermented with various bacterial combinations, including probiotic cultures. Fingerprinting was performed on samples collected at each hour of fermentation. Predictive modelling techniques, using both regression and classification approaches, were trialled in order to predict the change in signal intensity throughout fermentation. This aimed to understand if peptides could be predicted throughout fermentation, with a view to enable the targeted prediction of desirable peptides, or other relevant components, which may impart favourable sensory qualities in the final product. Regression techniques were somewhat effective for predicting the signal intensity of individual m/z ions throughout fermentation. Most of the ions did not follow a linear relationship, and, as such, a multiple linear regression model was unable to model most of the ions. Using a generalised additive model, a non-linear approach, improved the performance in most cases and could predict the signal intensity of individual ions throughout fermentation. However, the model was unable to correctly predict all cases. Classification techniques were effective for predicting the general direction of the signal intensity between start and end fermentation times. Five classification techniques were trialled, with each model providing accurate predictions for the increase or decrease of signal intensity between early and late fermentation times. Lastly, consumer panellists were recruited to evaluate the change in important sensory characteristics throughout the fermentation of milk prepared using two different starter cultures. This aimed to understand if consumer responses to such products could be correlated with instrumental analysis, in order to predict the consumer responses from instrumental data. Consumers perceived significant differences in bitterness and flavour intensity between fermented milk samples at different fermentation time points. There were significant correlations between peptide fingerprints and the consumer rankings for the sensory attributes in each fermented milk product. XGBoost regression could predict consumer responses with reasonable accuracy. This thesis explored the fermentation of milk using specific bacteria and fermentation processes. To validate this work, further products could be explored, in addition to different processing parameters. Furthermore, a more in-depth analysis of the chemical components of the products could be investigated and analysed with additional sensory evaluation to further explore and confirm the findings.Item The synthesis and chemistry of [2.2]paracyclophane amino acid derivatives : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatū, New Zealand(Massey University, 2020) Etheridge, LeonieDue to the ever-growing requirement for chiral compounds, new conditions for stereoselective synthesis are in constant development. Asymmetric organocatalysis is well-studied, with peptide catalysts popular due to their modular and highlyfunctionalisable nature. One such example of their utility is in the Michael reaction, a well-studied carbon-carbon bond forming reaction. [2.2]Paracyclophane is an aromatic industrial precursor compound with remarkable structural and electronic properties. Its conformational bulk and rigidity make it an attractive target for integration into sterically-hindered unnatural amino acids for incorporation into peptides that may be effective organocatalysts. An updated route to 4-amino-13-[2.2]paracyclophane-carboxylic acid (Pca) was developed and optimised. The synthetic route comprises four steps with an overall yield of 50%. This compares with previous routes which had yields between 7 and 48% for 6 - 7 steps. Peptide coupling conditions for the poorly-reactive Pca were developed with some success; including devising a route for direct synthesis of a glycine residue on Pca’s aniline. Four new Pca-containing peptides were described. The above work sets the stage for development of interesting new planar chiral peptide compounds with diverse chemistry. Three Pca-containing peptides were studied as asymmetric organocatalysts in Michael addition between trans-β-nitrostyrene and hexanal and were compared to proline, a known catalyst for this reaction. These tests were performed to probe the relationship between relative conformation between the carboxylic acid and amine moieties of the catalyst, and the catalyst’s stereoselectivity. The Pca-containing catalysts showed an interesting trend to reversal of the prevailing syn product configuration.Item Heterologous production and characterisation of a yeast peptide:N-glycanase : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, New Zealand(Massey University, 2007) Hong, KunPeptide:.N-glycanases (PNGases) removes A-linked glycans from glycoproteins. Three distinct families of PNGases have been characterised, although all of them not completely. Some of these PNGases are cytosolic, others are secreted. Cytoplasmic PNGases (Png lp) are implicated in the proteasomal degradation of newly synthesized misfolded or unfolded glycoproteins that are exported from the endoplasmic reticulum (ER). Cytoplasmic PNGases are encoded by the PNG1 gene and have been classified as members of transglutaminase-like superfamily based on the sequence analyses. There has, however, been no report of transglutaminase activity in any PNGase. The three-dimensional structures of recombinant PNGases from yeast (S. cerevisiae) and mouse have been determined in complex with the XPCB domain of Rad23 and mHR23B respectively. These PNGases were both produced as insoluble proteins, and could only be refolded and crystallised in the presence of their physiological binding partners. In this study, the gene encoding for S. pombe PNGase has been cloned and heterologously expressed as a soluble thioredoxin-fused protein. The proteolytic cleaved recombinant protein (rPNGase Sp) remained soluble as a monomer and retained its deglycosylating activity. It did not have, however any transglutaminase activity despite its homology to the transglutaminase family of proteins. The activity of rPNGase Sp in vitro is both reductant and Zn 2+- dependent. rP Gase Sp showed apparent heterogeneity on SDS-PAG E, which was characterised by the appearance of two bands differing in their molecular weights by an - 2.3 kDa. This heterogeneity was eventually shown to be the result of two different local conformations that were dependent on disulfide bond and/or Zn2 +. The enzyme was shown to only deglycosylatc the denatured glycoprotcins, not their native counterparts. Moreover, it preferred to dcglycosylatc glycoprotcins with high mannosc- typc glycan chains, both of which arc consistent with the biological function of cytoplasmic PNGascs. Compared to bacterial PNGasc F, rPNGasc Sp is not very active, at least on the substrate used in this study. A higher Km ( 186 μM) determined for rPNGasc Sp using a FITClabcllcd glycopcptidc which carries a complex-type glycan as the substrate also suggests that complex glycans arc not favoured substrates for these PNGascs. rPNGascSp has similar characteristics to the yeast (S. cerevisiae) and mouse PNGascs; it has a neutral pH optimum and is strongly inhibited by Cu2 +, Cd2 + and N j2+_ EDT A treatment deactivates it, and the addition of Zn2 + could not restore its activity. Interestingly, addition of exogenous Zn2 + was found to strongly inhibit rPNGasc Sp. dependent. rPNGase Sp showed apparent heterogeneity on SDS-PAGE, which was characterised by the appearance of two bands differing in their molecular weights by an ~ 2.3 kDa. This heterogeneity was eventually shown to be the result of two different local conformations that were dependent on disulfide bond and/or Zn 2+ . The enzyme was shown to only deglycosylate the denatured glycoproteins, not their native counterparts. Moreover, it preferred to deglycosylate glycoproteins with high mannose-type glycan chains, both of which are consistent with the biological function of cytoplasmic PNGases. Compared to bacterial PNGase F. rPNGase Sp is not very active, at least on the substrate used in this study. A higher K m (186 µM) determined for rPNGase Sp using a FITC-labelled glycopeptide which carries a complex-type glycan as the substrate also suggests that complex glycans are not favoured substrates for these PNGases. rPNGaseSp has similar characteristics to the yeast (S. cerevisiae) and mouse PNGases; it has a neutral pH optimum and is strongly inhibited by Cu 2+ , Cd 2+ and Ni 2+ EDTA treatment deactivates it, and the addition of Zn 2+ could not restore its activity. Interestingly, addition of exogenous Zn 2+ was found to strongly inhibit rPNGase Sp.Item Gastrointestinal endogenous proteins as a source of bioactive peptides : Doctor of Philosophy in Nutritional Sciences at Riddet Institute, Massey University, Palmerston North, New Zealand(Massey University, 2016) Acharya, PrasannalakshmiGastrointestinal endogenous proteins (GEP) were investigated as a source of bioactive peptides. In silico and in vitro methods were used singly or in combination to study GEP-derived peptides after simulated digestion. The presence of bioactive peptides after in vivo digestion was determined using a porcine model. Bioactivity of the peptides was assessed using selected in vitro bioactivity assays, and peptides were characterised using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. In the in silico study, twenty six different GEP and seven dietary proteins were subjected to simulated in silico gastrointestinal (SIGIT) digestion. The predicted resultant peptides possessing amino acid sequences identical to those of known bioactive peptides were identified by screening them against an online database of bioactive peptides (BIOPEP). The predicted number of bioactive peptides released after the SIGIT digestion of GEP ranged from 1 (secretin) to 39 (mucin-5AC), while those for dietary proteins ranged from 1 (gliadin) to 55 (myosin). Angiotensin-I-converting enzyme (ACE-I) inhibitory peptide sequences were found in abundance in both GEP and dietary proteins. The GEP mucin-5AC and the dietary protein myosin were predicted to release the highest number of ACE-I inhibitory peptides (38 and 49 peptides respectively), and were found to be comparable in their potential to release ACE-I inhibitory peptides. Following SIGIT digestion of eleven representative GEP, nineteen novel GEP-derived peptide sequences were selected by applying quantitative structure-activity relationship rules, and were chemically synthesised. Two novel peptides with the amino acid sequences RPCF and MIM, showing dipeptidyl peptidase IV (DPP-IV) inhibitory activity and five novel antioxidant (2,2-diphenyl-1-picrylhydrazyl (DPPH)- inhibitory and, or ferric reducing antioxidant power (FRAP) activity) peptides with amino acid sequences CCK, RPCF, CRPK, QQCP and DCR were identified. These results indicate that GEP may contain novel bioactive peptide sequences. The potential release of bioactive peptides, from four GEP (trypsin, lysozyme, mucin, and serum albumin) and a dietary protein (chicken albumin), in the gastrointestinal tract (GIT) was investigated using an in vitro digestion model. The in vitro digests were screened for ACE-I-, renin-, platelet-activating factor acetylhydrolase (PAF-AH)-, and DPP-IV-inhibition, and antioxidant activity. All four in vitro GEP digests showed ACE-I inhibition comparable to that of the positive control captopril. In comparison to the unfractionated digests, the enriched fractions (<3 and <10 kDa) of lysozyme and serum albumin showed greater renin-, PAF-AH-, and DPP-IV-inhibition, and antioxidant potential. Over 190 peptide sequences were identified from these fractions using mass spectrometry. Stomach chyme (SC) and jejunal digesta (JD) were collected from growing pigs that were fed a protein-free diet for a period of 3 days. The peptides extracted from SC and JD samples were characterized by SDS-PAGE, and their ACE-I-, DPPH-, and microsomal lipid peroxidation (MLP)- inhibition, FRAP activity determined. Potential bioactive peptides responsible for bioactivity were identified using mass spectrometry. SDS-PAGE analysis showed that all of the samples contained a heterogeneous mixture of peptides. Porcine JD samples inhibited ACE-I and DPPH, while SC samples inhibited MLP. Characterization studies identified over 180 peptide sequences from the enriched fractions of SC and JD samples that showed the highest activity. Further, a porcine serum albumin peptide sequence (FAKTCVADESAENCDKS) was found to be a sub-sequence of a larger sequence identified in the in vitro digest of human serum albumin. There was considerable inter-animal variation for the bioactivities. This may be attributed to sampling effects and, or natural variations in the gut contents, thus underlining the complexity involved in in vivo release of bioactive peptides. Together, the results indicate: 1) GEP contain abundant encrypted bioactive peptide sequences; 2) GEP-derived bioactive peptides display a range of bioactivities; 3) GEP-derived bioactive peptides are released during gastrointestinal digestion in pigs; 4) GEP may contain numerous novel bioactive peptide sequences encoded within their primary sequence. In conclusion, the evidence reported here suggests that, like the dietary proteins, GEP are also a potentially rich source of exogenously-derived bioactive peptides in the gastrointestinal tract. Beyond their primary functions, GEP may act as an important cryptomic source of bioactive peptides, given that the amount of GEP secreted into the gut is equal to or greater than the dietary protein ingested per day, and that up to 80% of GEP are known to be digested.Item Peptide sequences by mass spectrometry : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1970) Kent, Stephen Brian HenryThe preparation and mass spectrometry of permethylated peptide derivatives was investigated. Procedures for the modification of free peptides prior to permethylation were examined. Acetylation with methanol+ acetic anhydride was found to result in partial esterification of the peptide. Specific cleavage of the C-terminal residue was also observed; a mechanism is proposed for this reaction. Esterification with HCl in methanol followed by acetylation of the peptide ester gave a mixture of products due to random methanolysis during the esterification. Methods of acetylating free peptides were examined, and it was found that the use of water + acetic anhydride at room temperature resulted in rapid quantitative acetylation, with no significant side reactions. Reaction of an ethereal solution of diazomethane with the acetyl-peptide gave quantitative esterification with negligible byproduct formation. Use of dimethylsulfinyl sodium in dimethylsulfoxide, and methyl iodide for the permethylation of peptide derivatives was investigated. Suitable conditions were found for the preparation of the reagent and for its use in the permethylation reaction. Substitution at existing ester groups was found to occur during the permethylation, and the products were partially characterised. Use of the free acetyl-peptide rather than its methyl ester eliminated this side reaction. Introduction of more than the expected number of methyl groups was observed. This extra-methylation was found to occur mainly at specific residues, although some random methylation was observed. The conditions of permethylation were adjusted to minimise extra-methylation and limit it to specific sites in the molecule. Peptides containing aspartyl residues undergo chain cleavage; the products of this reaction were identified and a mechanism proposed for their formation. The permethylation reaction is discussed in relation to the formation of these artefacts; it is thought to involve deprotonation of the peptide to form a multiple anion. Reaction conditions are suggested to eliminate these side reactions. The mass spectrometry of permethylated peptide derivatives is discussed and the mass spectra of peptides of known sequence reported. The mass spectra show the sequence-determining fragments as the principal ions. This observation is rationalised in terms of the negative-inductive effect of the N-methyl groups. The simple procedure for interpreting the mass spectra of permethylated peptide derivatives is outlined, together with the use of minor fragmentation modes in identifying the molecular ion and sequencing peaks. Deuteriated methyl iodide, high resolution mass spectrometry and the detection of metastable transitions can all be used to confirm the deduced sequence. The techniques developed were applied to a mixture of free peptides isolated from cheese; the three peptides present were sequenced. The results were confirmed by high resolution mass measurement and permethylation with deuteriated methyl iodide. The present state of peptide sequence determination by mass spectometry is evaluated and possible future developments discussed.Item The amino acid sequence of tryptic peptides of sheep heart phosphofructokinase : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University(Massey University, 1974) Brennan, Stephen OliverPhosphofructokinase was purified from sheep heart. The sedimentation pattern of the purified enzyme was investigated over a protein concentration range 0.7 to 14.5 mg/ml. Two distinct 7 and 30 S boundaries were observed at all concentrations. A minor amount of 19 S material was also present. The 30 S boundary was asymmetric and its concentration dependence was characteristic of a polymerising system in rapid reversible equilibrium. The dissolved crystalline enzyme usually sedimented as a single trailing 30 S boundary; the molecular weight of this component was estimated at 1.5 x 10 6. This value was consistent with x-ray data, which indicated unit cell dimensions of 600 x 250 x 220 Å, implying a protein weight greater than 106 daltons per asymmetric unit. In one experiment the 30 S component appeared to be undergoing a trimerisation to a 53 S form. Sodium dodecylsulphate gel electrophoresis indicated a protomer molecular weight of 80,000 to 85,000, which was consistent with a corrected sedimentation coefficient of 3.8 S and a molecular weight of 90,000 for maleyl-phosphofructokinase, and with a corrected sedimentation coefficient of 3.9 S for the urea-dissociated enzyme. When maleylation was carried out on carboxymethyl-phosphofructokinase in 7.5 M urea, the enzyme was further dissociated to a 40,000 molecular weight subunit. Peptide mapping of tryptic peptides; in which arginine-, histidine-, tryptophan- and tyrosine-containing peptides were located; was consistent with an 85,000 form composed of two identical subunits. The enzyme was digested with trypsin. Forty-three different peptides were isolated using a combination of: gel filtration, ion exchange chromatography (on Dowex 50 and DE 32 cellulose), paper electrophoresis, and paper chromatography. The complete amino acid sequence was established for 38 of these peptides. The amino acid compositions (and partial sequences) were established for the other five tryptic peptides. A summary of the amino acid sequence data obtained for the tryptic peptides is shown in Table V. Seven carboxymethylcysteine-containing peptides were isolated in this investigation, while eight have been isolated from rabbit muscle (Coffee et al. 1973). Six of these peptides had very similar compositions between the two species. The rabbit enzyme contained two carboxymethylcysteine-containing peptides which were not found in sheep heart and the sheep enzyme contained a 20 residue peptide not found in rabbit muscle. This probably reflects genetic variation between the two species.