Isolation and characterization of Bradykinin potentiating peptides from Agkistrodon bilineatus venom
© Munawar et al. 2016
Received: 9 July 2015
Accepted: 5 January 2016
Published: 14 January 2016
Snake venom is a source of many pharmacologically important molecules. Agkistrodon bilineatus commonly known as Cantil, is spread over Central America particularly in Mexico and Costa Rica. From the venom of Agkistrodon bilineatus we have isolated and characterised six hypotensive peptides, and two bradykinin inhibitor peptides. The IC-50 value of four synthesized peptides was studied, towards angiotensin converting enzyme, in order to study the structure-function relationship of these peptides.
The purification of the peptides was carried out by size exclusion chromatography, followed by reverse phase chromatography. Sequences of all peptides were determined applying MALDI-TOF/TOF mass spectrometry. These hypotensive peptides bear homology to bradykinin potentiating peptides and venom vasodilator peptide. The peptide with m/z 1355.53 (M + H)+1, and the corresponding sequence ZQWAQGRAPHPP, we identified for the first time. A precursor protein containing a fragment of this peptide was reported at genome level, (Uniprot ID P68515), in Bothrops insularis venom gland. These proline rich hypotensive peptides or bradykinin potentiating peptides are usually present in the venom of Crotalinae, and exhibit specificity in binding to the C domain of somatic angiotensin converting enzyme. Four of these hypotensive peptides, were selected and synthesized to obtain the required quantity to study their IC50 values in complex with the angiotensin converting enzyme. The peptide with the sequence ZLWPRPQIPP displayed the lowest IC50 value of 0.64 μM. The IC50 value of the peptide ZQWAQGRAPHPP was 3.63 μM.
The canonical snake venom BPPs classically display the IPP motif at the C-terminus. Our data suggest that the replacement of the highly conserved hydrophobic isoleucine by histidine does not affect the inhibitory activity, indicating that isoleucine is not mandatory to inhibit the angiotensin converting enzyme. The evaluation of IC 50 values show that the peptide with basic pI value exhibits a lower IC 50 value.
KeywordsSnake venom Peptides Angiotensin converting enzyme Hypotension MALDI/TOF-TOF Size exclusion chromatography
Snake venom is known for its toxic and lethal effects in its prey. Nature has endowed this creature, with this special secretion to survive in a particular niche . Snake venoms consists of enzymatic and non enzymatic proteinaceous components, which can be grouped into several families based on their structural and functional relationship . Although the members of a single family show remarkable similarities in their primary, secondary and tertiary structures, they often exhibit distinct different pharmacological effects . Studies are being carried not only to unravel and characterize the composition of snake venom, but also to identify and develop novel therapeutics from venoms for the benefit of mankind [3–8]. Particularly venom peptides are today of great interest in this regard, because as a result of the evolutionary process, they have attained highly stable molecular scaffolds, which are resistant to degradation by proteases. Beside these peptides are poorly immunogenic they can be easily synthesized. The structural stability of some venom peptides is a result of disulfide bond formation and posttranslational modification . The most common type of the posttranslational modification observed in snake venom peptides is a pyroglutamate residue at the N-terminus . Bradykinin potentiating peptides (BPP) are a good example in this respect. These peptides were isolated from the venom of Bothrops jararaca . Captopril, which was the first orally active inhibitor of the angiotensin converting enzyme was designed based on the structure of BPPs isolated from Bothhrops jararaca venom . Since then numerous studies have been made to isolate and characterize BPPs from different snake venoms [13–18]. Efforts are being made to study the structure function relationship of this type of peptides in detail, and their possible modes of blood pressure lowering or vasodilatation. For example it was shown that the peptide Bj-PRO-10c  inhibits ACE, however argininosuccinate synthetase, present in the kidney cytosol is its primary target [15, 19–21].
Here we summarize the isolation and characterisation of five bradykinin potentiating peptides, one vasodilator peptide and three bradykinin inhibiting peptides from the venom of Agkistrodon bilineatus, generally known as Cantil. In order to determine their IC 50 values, the inhibitory activities of the synthetic analogues of four natural BPPs peptides towards angiotensin converting enzymes were also studied.
Isolation of Bradykinin potentiating peptides from Agkistrodon bilineatus venom
Peptides identified in Agkistrodon bilineatus venom
Observed Mass (M + H)+
Homology with the peptide
Q27J49: Lachesis muta muta
BPP and CNP
P85025: Agkistrodon bilineatus
BPP and CNP
P0C7K3: Crotalus viridis viridis
Q7T1M3: Bothrops jararacussu
BPP and CNP
P0C7S6: Crotalus atrox
P68515: Bothrops insularis
BPP and CNP
P0C7R7: Bothrops alternatus
P84746: Bothrops jararacussu
venom vasodilator peptide
The peptide sequencing was done in an automated mode, by applying Mascot Inhouse search. Although with MALDI-TOF/TOF, it is not possible to differentiate between isobaric leucine and isoleucine, and quasi-isobaric (Gln/Lys) residues, however previous studies, Edman degradation and cDNA, of similar BPPs, support the presence of the amino acid isoleucine instead of leucine, and Gln rather than Lys [16, 22–24]. Therefore at all places in the manuscript X refers to Ile/Leu.
Determination of IC 50 values
IC 50 values of the synthetic analogues of natural BPPs for the angiotensin converting enzyme
Calculated pI value
IC 50 (μM)
Inhibitor 1 (P6)
Inhibitor 2 (P7)
Inhibitor 3 (P5)
Inhibitor 4 (P3)
Snake venoms, are a source of many molecules with biomedical importance. The BPPs isolated and characterized within this work show homology with BPPs already isolated from other snake venoms. However, an extensive data base search revealed that peptide 7 with the sequence ZQWAQGRAPHPP has been identified for the first time. All previous descriptions of this peptide were inferred from transcripts, with no evidence of secretion of the gene product in the venom. The analysis of IC 50 values (Table 2) for ACE, applying four synthetic replica of these BPPs, provided further insights into the structure-function relationship of these peptides. The low IC 50 value of the synthetic peptide ZQWAQGRAPHPP indicates that the presence isoleucine in the IPP sequence is not mandatory to inhibit ACE. Furthermore the calculated pI values of these peptides suggest that the peptides with basic pI value show a lower IC 50 value. Secondly the sequence analysis of the BPPs isolated and described in this work and other studies [13, 14, 18, 34], show that these peptides usually have a pyroglumate modification at their N-terminus, and have a high ratio of proline residues. These two factors provide inherent stability to these peptides towards degradation, particularly in venom glands, which contain various types of enzymes, as well as in the prey's blood. Further, it can be concluded that the presence of peptide 2 (bradykinin inhibitor peptide) reinforces the idea that these peptides are conserved in the venom of Agkistrodon bilineatus over the process of evolution and that these molecules, so far found in many venoms, must play a key role in predation.
The snake venom of Agkistrodon bilineatus bilineatus, was obtained from Venom Supplies, Australia, and was placed at -20° till further use. The venom was collected from the snakes bred in the farms of Venom Supplies, Australia. All the solvents used were of HPLC grade and were obtained from Merck. The substrate Abz-Phe-Arg-Lys (Dnp)-Pro-OH for angiotensin converting enzyme was obtained from BACHEM. Angiotensin converting enzyme from rabbit lung (A6778- 0.25 UN), was obtained from Sigma. The synthesized peptides ZLWPRPQIPP, ZQWAQGRAPHPP, ZSAPGNEAIPP, ZNWPHPQIPP were purchased from China Peptide company, Shanghai, China. The peptides were 95 % pure, and were kept at -20° till use. The N-terminus of these synthetic peptide is pyroglutame (represented by Z), while the C-terminus is an amino acid with free carboxylic group.
Purification of Bradykinin potentiating peptides (BPP)
The crude venom (50 mg) was dissolved in 1 ml of 100 mM ammonium acetate (pH 5), and centrifuged at 13000 rps. The undissolved material settled down, and the supernatant of crude venom solution was fractionated using a size exclusion column (Superdex-75, 16 x 60 mm) connected to an ÄKTA Purifier system (GE Healthcare). 100 mM ammonium acetate (pH 5) was used as the elution buffer. The fractionation was performed at a rate of 1 ml/min, and UV absorbance of the eluate was monitored at 220 and 280 nm. Fractions were collected and subjected to SDS-PAGE (15 % glycine gels) under non reducing conditions. The gels were stained with Coomassie Blue. The peptide fractions (peak 8-9, Fig. 1) showing inhibitory activity towards the angiotensin converting enzyme were filtered through 3 kDa Amicon filter and further purified with Chromolith-C-18 column (100x 4.6 mm), using an Agilent 1200 system. Solvent A was 0.2 % formic acid in water and B was straight acetonitrile. A stepwise gradient 3-30 % B for 28 min, 30-40 % B for 9 min and 40-60 % B for 3 min and a flow rate of 2 ml/min was applied to isolate the peptides (Fig. 2). UV absorbance was monitored at 220 nm and 280 nm.
Enzyme inhibition assay
ACE activities in the presence of venom peptides were determined by a fluorescence energy transfer assay using Abz-Phe-Arg-Lys (Dnp)-Pro-OH as a substrate . 1 mg of the substrate was weighed and dissolved in 1 ml DMSO. The exact concentration of the substrate was determined by taking four different volumes of the substrate stock solution, and constructing a standard curve spectrophometrically at 365 nm, using the molar extinction coefficient of the Dnp group (ε356 = 17,300 M−1 cm−1), according to the Beer’s Lambert Law (A = εDnp x l x c). A stock solution of the enzyme was prepared by suspending 0.25 UN of ACE in 250 μl of the assay buffer (12.10 g Tris-base, 2.92 g NaCl and 1.36 mg ZnCl2 in 1 l of deionised water, pH to 7.0 adjusted with HCl).
In order to determine the IC 50 values of the synthesized peptides, a stock solution (10 mM) of each was prepared in the assay buffer. From the stock solutions, six serial dilutions were prepared for each peptide. The concentrations of the six dilutions were as follows, 0.500 mM, 0.167 mM, 0.056 mM, 0.019 mM, 0.006 mM and 0.002 mM respectively. In the final assay, 85 μl of the assay buffer, 2 μl of the enzyme stock solution, and 5 μl of the dilute peptide solution were added. The mixture was incubated for 10 min at room temperature. The reaction was started by adding 10 μl of the working solution of the substrate (20 μl of the stock +280 μl of the assay buffer). The same procedure was adopted using 5 μl of venom fractions instead of the synthetic peptides. Fluorescence measurements were made at λex = 320 nm and at λem = 420 nm, for 5 min each. These experiments were repeated five times to ensure accuracy of the results.
Matrix-assisted desorption/ionization time-of-flight mass spectrometry
MALDI-TOF-TOF analyses were performed with a ultrafleXtreme instrument (Bruker Daltonics, Bremen, Germany). Samples were dried after reversed phase chromatography, dissolved in 30 % ACN, 0.1 % TFA in H2O and 1 μl of the solution was spotted on a MALDI target plate (MTP AnchorChip 384, Bruker Daltonics). After drying 1 μl MALDI matrix (0.7 mg/ml Cyano-4-hydroxycinnamic acid (Bruker Daltonics) dissolved in 85 % ACN, 1 mM NH4H2PO4 and 0.1 % TFA dissolved in H2O) were spotted on the sample plate.
Data acquisition was performed in positive ion mode using the flexControl software 3.3. The parameters were set as follows: ion source 1: 25 kV, ion source 2: 23.6 kV, lens: 7.5 kV. MS data were collected automatically using autoXecute. Parameters were set as follows: laser power: 47 %; laser shots: 1000; movement, random walk with 100 shots per raster spot. Peaks were selected for LIFT measurement if they met the following criteria: signal to noise > 8, peak intensity > 300.
MS spectra were processed applying flexAnalysis (version 3.3, Bruker Daltonics). Further data analysis was performed using BioTools (version3.2, Bruker Daltonics) and Mascot Inhouse Search. Mascot version 2.1.03 was used to analyse and search the spectra against the subset “other lobe-finned fish and tetrapod clade” of the Swissprot database. The precursor ion mass tolerance was set to 1 Da, the fragment ion mass tolerance was 0.5 Da.
angiotensin converting enzyme
bradykinin potentiating peptides
AM acknowledges the University of Hamburg, Germany for supporting a research visit. C.B., A.M. and PS acknowledge support by DAAD via PROBAL and support from CNPq, Brazil under project number 33.654.831/1001-36. AM and AZ acknowledge UET, Lahore, Pakistan for support, via Faculty Research Project No. ORIC/94-ASRB/143.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Meier J, Stocker KF. Biology and distributuion of venomous snakes of medical importance and the composition of snake venom. In: Meier JWJ, editor. Handbook of clinical toxicology of animal venoms and proteins. Boca Raton, Florida: CRC Press; 1995. p. 367–412.Google Scholar
- Georgieva DAR, Betzel C. Proteome analysis of snake venom toxins: pharmacological insights. Expert Rev Proteomics. 2008;5(6):787–97.PubMedView ArticleGoogle Scholar
- Kang TS, Georgieva D, Genov N, Murakami MT, Sinha M, Kumar RP, et al. Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis. FEBS J. 2011;278(23):4544–76.PubMedView ArticleGoogle Scholar
- Wang Y, Hong J, Liu X, Yang H, Liu R, Wu J, et al. Snake cathelicidin from Bungarus fasciatus is a potent peptide antibiotics. PLoS One. 2008;3(9):e3217.PubMedPubMed CentralView ArticleGoogle Scholar
- Calvete JJ. Venomics: digging into the evolution of venomous systems and learning to twist nature to fight pathology. J Proteome. 2009;72(2):121–6.View ArticleGoogle Scholar
- Sanchez EE, Rodriguez-Acosta A, Palomar R, Lucena SE, Bashir S, Soto JG, et al. Colombistatin: a disintegrin isolated from the venom of the South American snake (Bothrops colombiensis) that effectively inhibits platelet aggregation and SK-Mel-28 cell adhesion. Arch Toxicol. 2009;83(3):271–9.PubMedView ArticleGoogle Scholar
- Zhang Y, Wu J, Yu G, Chen Z, Zhou X, Zhu S, et al. A novel natriuretic peptide from the cobra venom. Toxicon. 2011;57(1):134–40.PubMedView ArticleGoogle Scholar
- Earl ST, Masci PP, de Jersey J, Lavin MF, Dixon J. Drug development from Australian elapid snake venoms and the Venomics pipeline of candidates for haemostasis: Textilinin-1 (Q8008), Haempatch (Q8009) and CoVase (V0801). Toxicon. 2012;59(4):456–63.PubMedView ArticleGoogle Scholar
- Pimenta AM, De Lima ME. Small peptides, big world: biotechnological potential in neglected bioactive peptides from arthropod venoms. J Pept Sci. 2005;11(11):670–6.PubMedView ArticleGoogle Scholar
- Munawar A, Trusch M, Georgieva D, Spencer P, Frochaux V, Harder S, et al. Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae. Mol BioSyst. 2011;7(12):3298–307.PubMedView ArticleGoogle Scholar
- Ondetti MA, Williams NJ, Sabo EF, Pluscec J, Weaver ER, Kocy O. Angiotensin-converting enzyme inhibitors from the venom of Bothrops jararaca. Isolation, elucidation of structure, and synthesis. Biochemistry. 1971;10(22):4033–9.PubMedView ArticleGoogle Scholar
- Cushman DW, Ondetti MA. Design of angiotensin converting enzyme inhibitors. Nat Med. 1999;5(10):1110–3.PubMedView ArticleGoogle Scholar
- Hayashi MA, Camargo AC. The Bradykinin-potentiating peptides from venom gland and brain of Bothrops jararaca contain highly site specific inhibitors of the somatic angiotensin-converting enzyme. Toxicon. 2005;45(8):1163–70.PubMedView ArticleGoogle Scholar
- Wermelinger LS, Dutra DL, Oliveira-Carvalho AL, Soares MR, Bloch Jr C, Zingali RB. Fast analysis of low molecular mass compounds present in snake venom: identification of ten new pyroglutamate-containing peptides. Rapid Commun Mass Spectrom. 2005;19(12):1703–8.PubMedView ArticleGoogle Scholar
- Camargo AC, Ianzer D, Guerreiro JR, Serrano SM. Bradykinin-potentiating peptides: beyond captopril. Toxicon. 2012;59(4):516–23.PubMedView ArticleGoogle Scholar
- Lopes DM, Junior NE, Costa PP, Martins PL, Santos CF, Carvalho ED, et al. A new structurally atypical bradykinin-potentiating peptide isolated from Crotalus durissus cascavella venom (South American rattlesnake). Toxicon. 2014;90:36–44.PubMedView ArticleGoogle Scholar
- Munawar A, Trusch M, Georgieva D, Hildebrand D, Kwiatkowski M, Behnken H, et al. Elapid snake venom analyses show the specificity of the peptide composition at the level of genera Naja and Notechis. Toxins. 2014;6(3):850–68.PubMedPubMed CentralView ArticleGoogle Scholar
- Rioli V, Prezoto BC, Konno K, Melo RL, Klitzke CF, Ferro ES, et al. A novel bradykinin potentiating peptide isolated from Bothrops jararacussu venom using catallytically inactive oligopeptidase EP24.15. FEBS J. 2008;275(10):2442–54.PubMedView ArticleGoogle Scholar
- Guerreiro JR, Lameu C, Oliveira EF, Klitzke CF, Melo RL, Linares E, et al. Argininosuccinate synthetase is a functional target for a snake venom anti-hypertensive peptide: role in arginine and nitric oxide production. J Biol Chem. 2009;284(30):20022–33.PubMedPubMed CentralView ArticleGoogle Scholar
- Morais KL, Hayashi MA, Bruni FM, Lopes-Ferreira M, Camargo AC, Ulrich H, et al. Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B(2)and M1 muscarinic acetylcholine receptors. Biochem Pharmacol. 2011;81(6):736–42.PubMedView ArticleGoogle Scholar
- Paschoal JF, Yamaguchi J, Miranda JR, Carretero G, Melo RL, Santos RA, et al. Insights into cardiovascular effects of proline-rich oligopeptide (Bj-PRO-10c) revealed by structure-activity analyses: dissociation of antihypertensive and bradycardic effects. Amino Acids. 2014;46(2):401–13.PubMedView ArticleGoogle Scholar
- Faria Ferreira LA, Galle MR A, Schrader M, Lebrun I, Habermehl G. Isolation: Analysis and Properties of Three Brady kinin-Potentiating Peptides (BPP-II, BPP-III, and BPP-V) From Bothrops Neuwiedi Venom. J Protein Chem. 1998;17(3):285–9.View ArticleGoogle Scholar
- Gomes CL, Konno K, Conceição IM, Ianzer D, Yamanouye N, Prezoto BC, et al. Identification of novel bradykinin-potentiating peptides(BPPs) in the venom gland of a rattlesnake allowed the evaluation of the structure–function relationship of BPPs. Biochem Pharmacol. 2007;74(9):1350–60.PubMedView ArticleGoogle Scholar
- Kodama RT, Carvalho DC, Kuniyoshi AK, Kitano ES, Tashima AK, Barna BF et al. New proline-rich oligopeptides from the venom of African Adders: Insights into the hypotensive effect of the venoms. Biochimica et biophysica acta. 2015; doi:10.1016/j.bbagen.2015.02.005
- Majumder K, Wu J. Molecular Targets of Antihypertensive Peptides: Understanding the Mechanisms of Action Based on the Pathophysiology of Hypertension. Int J Mol Sci. 2014;16(1):256–83.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhuo JL, Ferrao FM, Zheng Y, Li XC. New frontiers in the intrarenal Renin-Angiotensin system: a critical review of classical and new paradigms. Front Endocrinol. 2013;4:166.View ArticleGoogle Scholar
- Junqueira-de-Azevedo Ide L, Ho PL. A survey of gene expression and diversity in the venom glands of the pitviper snake Bothrops insularis through the generation of expressed sequence tags (ESTs). Gene. 2002;299(1-2):279–91.PubMedView ArticleGoogle Scholar
- Murayama N, Hayashi MA, Ohi H, Ferreira LA, Hermann VV, Saito H, et al. Cloning and sequence analysis of a Bothrops jararaca cDNA encoding a precursor of seven bradykinin-potentiating peptides and a C-type natriuretic peptide. Proc Natl Acad Sci U S A. 1997;94(4):1189–93.PubMedPubMed CentralView ArticleGoogle Scholar
- Cintra AC, Vieira CA, Giglio JR. Primary structure and biological activity of bradykinin potentiating peptides from Bothrops insularis snake venom. J Protein Chem. 1990;9(2):221–7.PubMedView ArticleGoogle Scholar
- Souza GH, Catharino RR, Ifa DR, Eberlin MN, Hyslop S. Peptide fingerprinting of snake venoms by direct infusion nano-electrospray ionization mass spectrometry: potential use in venom identification and taxonomy. J Mass Spectrom. 2008;43(5):594–9.PubMedView ArticleGoogle Scholar
- Cotton J, Hayashi MA, Cuniasse P, Vazeux G, Ianzer D, De Camargo AC, et al. Selective inhibition of the C-domain of angiotensin I converting enzyme by bradykinin potentiating peptides. Biochemistry. 2002;41(19):6065–71.PubMedView ArticleGoogle Scholar
- Lameu C, Ulrich H. Applications of Snake Venom Proline-Rich Oligopeptides (Bj- PROs) in Disease Conditions Resulting from Deficient Nitric Oxide Production. INTECH Open Access Publisher; Rijeka, Croatia 2013
- Masuyer G, Schwager SL, Sturrock ED, Isaac RE, Acharya KR. Molecular recognition and regulation of human angiotensin-I converting enzyme (ACE) activity by natural inhibitory peptides. Sci Rep. 2012;2:717.PubMedPubMed CentralView ArticleGoogle Scholar
- Ianzer D, Konno K, Marques-Porto R, Vieira Portaro FC, Stocklin R, de Camargo AC M, et al. Identification of five new bradykinin potentiating peptides (BPPs) from Bothrops jararaca crude venom by using electrospray ionization tandem mass spectrometry after a two-step liquid chromatography. Peptides. 2004;25(7):1085–92.PubMedView ArticleGoogle Scholar
- Coutinho-Neto ACC, Souza GH, Zaqueo KD, Kayano AM, Silva RS, Zuliani JP, et al. ESI-MS/MS identification of a bradykinin-potentiating peptide from Amazon Bothrops atrox snake venom using a hybrid Qq-oaTOF mass spectrometer. Toxins. 2013;5(2):327–35.PubMedPubMed CentralView ArticleGoogle Scholar
- Pina AS, Roque AC. Studies on the molecular recognition between bioactive peptides and angiotensin-converting enzyme. J Mol Recognit. 2009;22(2):162–8.PubMedView ArticleGoogle Scholar
- Graham RL, Graham C, McClean S, Chen T, O'Rourke M, Hirst D, et al. Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers. Biochem Biophys Res Commun. 2005;338:1587–92.PubMedView ArticleGoogle Scholar
- Lomonte B, Tsai WC, Ureña-Diaz JM, Sanz LM-OD, Sánchez EE, Fry BGGJ, et al. Venomics of New World pit vipers: genus-wide comparisons of venom proteomes across Agkistrodon. J Proteome. 2014;16(96):103–16.View ArticleGoogle Scholar
- Carmona AK, Schwager SL, Juliano MA, Juliano L, Sturrock ED. A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay. Nat Protoc. 2006;1(4):1971–6.PubMedView ArticleGoogle Scholar
- Tashima AK, Zelanis A, Kitano ES, Ianzer D, Melo RL, Rioli V, et al. Peptidomics of three Bothrops snake venoms: insights into the molecular diversification of proteomes and peptidomes. Mol Cell Proteomics. 2012;11(11):1245–62.PubMedPubMed CentralView ArticleGoogle Scholar
- Hayashi MA, Murbach AF, Ianzer D, Portaro FC, Prezoto BC, Fernandes BL, et al. The C-type natriuretic peptide precursor of snake brain contains highly specific inhibitors of the angiotensin-converting enzyme. J Neurochem. 2003;85(4):969–77.PubMedView ArticleGoogle Scholar