Specific Nitration at Tyrosine 430 Revealed by High Resolution Mass Spectrometry as Basis for Redox Regulation of Bovine Prostacyclin Synthase

Treatment of bovine aortic microsomes containing active prostacyclin synthase (PGI2 synthase) with increasing concentrations of peroxynitrite (PN) up to 250 μm of PN yielded specific staining of this enzyme on Western blots with antibodies against 3-nitrotyrosine (3-NT), whereas above 500 μm PN staining of additional proteins was also observed. Following treatment of aortic microsomes with 25 μm PN, PGI2 synthase was about half-maximally nitrated and about half-inhibited. It was then isolated by gel electrophoresis and subjected to proteolytic digestion with several proteases. Digestion with thermolysin for 24 h provided a single specific peptide that was isolated by high performance liquid chromatography and identified as a tetrapeptide Leu-Lys-Asn-Tyr(3-nitro)-COOH corresponding to positions 427–430 of PGI2 synthase. Its structure was established by precise mass determination using Fourier transform-ion cyclotron resonance-nanoelectrospray mass spectrometry and Edman microsequencing and ascertained by synthesis and mass spectrometric characterization of the authentic Tyr-nitrated peptide. Complete digestion by Pronase to 3-nitrotyrosine was obtained only after 72 h, suggesting that the nitrated Tyr-430 residue may be embedded in a tight fold around the heme binding site. These results provide evidence for the specific inhibition of PGI2 synthase by nitration at Tyr-430 that may occur already at low levels of PN as a consequence of endothelial co-generation of nitric oxide and superoxide. Treatment of bovine aortic microsomes containing active prostacyclin synthase (PGI2 synthase) with increasing concentrations of peroxynitrite (PN) up to 250 μm of PN yielded specific staining of this enzyme on Western blots with antibodies against 3-nitrotyrosine (3-NT), whereas above 500 μm PN staining of additional proteins was also observed. Following treatment of aortic microsomes with 25 μm PN, PGI2 synthase was about half-maximally nitrated and about half-inhibited. It was then isolated by gel electrophoresis and subjected to proteolytic digestion with several proteases. Digestion with thermolysin for 24 h provided a single specific peptide that was isolated by high performance liquid chromatography and identified as a tetrapeptide Leu-Lys-Asn-Tyr(3-nitro)-COOH corresponding to positions 427–430 of PGI2 synthase. Its structure was established by precise mass determination using Fourier transform-ion cyclotron resonance-nanoelectrospray mass spectrometry and Edman microsequencing and ascertained by synthesis and mass spectrometric characterization of the authentic Tyr-nitrated peptide. Complete digestion by Pronase to 3-nitrotyrosine was obtained only after 72 h, suggesting that the nitrated Tyr-430 residue may be embedded in a tight fold around the heme binding site. These results provide evidence for the specific inhibition of PGI2 synthase by nitration at Tyr-430 that may occur already at low levels of PN as a consequence of endothelial co-generation of nitric oxide and superoxide. peroxynitrite (oxoperoxonitrate (1−)) prostacyclin prostacyclin synthase 3-nitrotyrosine prostaglandin endoperoxide 6-keto-prostaglandin F1α enzyme immunoassay nitric oxide phosphate-buffered saline bovine serum albumin bacterial monooxygenase-3 from Bacillus megaterium (CYP 102) Fourier transform ion cyclotron resonance high performance liquid chromatography phenylthiohydantoin matrix-assisted laser desorption ionization time-of-flight N-(9-fluorenyl)methoxycarbonyl The nitration of tyrosine residues in proteins has become a well recognized reaction, but has been heavily disputed with regard to the mechanisms involved and its physiological and/or pathophysiological significance (1Cohen R.A. Prog. Cardiovasc. Dis. 1995; 38: 105-128Crossref PubMed Scopus (261) Google Scholar, 2Moncada S. Vane J.R. Pharmacol. Rev. 1979; 30: 293-331Google Scholar, 3Moncada S. Vane J.R. N. Engl. J. Med. 1979; 300: 1142-1147Crossref PubMed Scopus (914) Google Scholar, 4Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar, 5Zou M.H. Jendral M. Ullrich V. Br. J. Pharmacol. 1999; 126: 1283-1292Crossref PubMed Scopus (83) Google Scholar). Peroxynitrite (PN)1 generated from nitric oxide (NO) and superoxide (O2⨪) can react with Tyr or Tyr-containing proteins under formation of 3-nitrotyrosine (3-NT) (6Crow J.P. Beckman J.S. Adv. Pharmacol. 1995; 34: 17-43Crossref PubMed Scopus (289) Google Scholar, 7Beckman J.S. Koppenol W.H. Am. J. Physiol. 1996; 271: C1424-C1437Crossref PubMed Google Scholar, 8Viner R.I. Williams T.D. Schöneich C. Biochemistry. 1999; 38: 12408-12415Crossref PubMed Scopus (211) Google Scholar) but in general the required concentrations are higher than expected to occur in vivo. Pfeiffer and Mayer (9Pfeiffer S. Mayer B. J. Biol. Chem. 1998; 273: 27280-27285Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 10Pfeiffer S. Schmidt K. Mayer B. J. Biol. Chem. 2000; 275: 6346-6352Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 11Pfeiffer S. Lass A. Schmidt K. Mayer B. J. Biol. Chem. 2001; 276: 34051-34058Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 12Pfeiffer S. Lass A. Schmidt K. Mayer B. FASEB J. 2001; 15: 2355-2364Crossref PubMed Scopus (100) Google Scholar) have even questioned the significance of PN as a cellular nitrating agent and have proposed nitrite/hydrogen peroxide as an alternative pathway with myeloperoxidase as a catalyst (11Pfeiffer S. Lass A. Schmidt K. Mayer B. J. Biol. Chem. 2001; 276: 34051-34058Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 12Pfeiffer S. Lass A. Schmidt K. Mayer B. FASEB J. 2001; 15: 2355-2364Crossref PubMed Scopus (100) Google Scholar), which may indeed apply for certain proteins. In the case of PN it has not been considered that PN can be activated by transition metal ions that may then catalyze the self-nitration of metalloproteins at low PN levels. We have recently provided evidence for this reaction for heme-thiolate (P450) proteins (13Daiber A. Schöneich C. Schmidt P. Jung C. Ullrich V. J. Inorg. Biochem. 2000; 81: 213-220Crossref PubMed Scopus (35) Google Scholar, 14Daiber A. Herold S. Schöneich C. Namgaladze D. Peterson J.A. Ullrich V. Eur. J. Biochem. 2000; 267: 6729-6739PubMed Google Scholar, 15Heinz K. Dünstl G. Bachschmid M. Daiber A. Nüsing R. Ullrich V. Nitric Oxide. 2002; 6: 400Google Scholar) that therefore may serve as a model for the P450 protein PGI2 synthase. PGI2 synthase was inactivated by micromolar PN concentrations (16Zou M.H. Ullrich V. FEBS Lett. 1996; 382: 101-104Crossref PubMed Scopus (198) Google Scholar, 17Zou M.H. Martin C. Ullrich V. Biol. Chem. 1997; 378: 707-713Crossref PubMed Scopus (270) Google Scholar) but also by a continuous generation of NO and O2⨪ from SIN-1 (18Zou M.H. Klein T. Pasquet J.-P. Ullrich V. J. Biochem. 1998; 336: 507-512Crossref Scopus (64) Google Scholar). In cellular systems the inhibition of nitration by a NO synthase inhibitor and polyethylene-glycolated superoxide dismutase provided evidence for the involvement of PN, whereas nitrite was ineffective (18Zou M.H. Klein T. Pasquet J.-P. Ullrich V. J. Biochem. 1998; 336: 507-512Crossref Scopus (64) Google Scholar). Because NO and PGI2 are important for the endothelial barrier function the formation of PN and the nitration of PGI2 synthase could play a role in the process of endothelial activation for adhesion and emigration of white blood cells into the tissue (19Ullrich V. Zou M.H. Bachschmid M. Biochim. Biophys. Acta. 2001; 1532: 1-14Crossref PubMed Scopus (30) Google Scholar). Interestingly, PGI2 synthase was found localized to the caveolae-like endothelial NO synthase (20Spisni E. Griffoni C. Santi S. Riccio M. Marulli R. Bartolini G. Toni M. Ullrich V. Tomasi V. Exp. Cell Res. 2001; 266: 31-43Crossref PubMed Scopus (72) Google Scholar) and hence PN formation may occur in close vicinity to PGI2 synthase. This localization in a “quasi-extracellular” compartment may be a further important factor for efficient nitration by low concentrations of PN. Beyond this physiological background no proof for the molecular basis of enzyme inhibition has been hitherto obtained by identification of nitrated tyrosine. Substrate analogs of prostaglandin-endoperoxide have been recently shown to inhibit the nitration (17Zou M.H. Martin C. Ullrich V. Biol. Chem. 1997; 378: 707-713Crossref PubMed Scopus (270) Google Scholar), which suggested a proximity to the heme attached to the protein by the Cys-441 residue (21Hatae T. Hara S. Yokoyama C. Yabuki T. Inoue H. Ullrich V. Tanabe T. FEBS Lett. 1996; 389: 268-272Crossref PubMed Scopus (34) Google Scholar, 22Shyue S. Ruan K. Wang L. Wu K. J. Biol. Chem. 1997; 272: 3657-3662Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Ullrich V. Brugger R. Angew. Chem. Int. Ed. Engl. 1994; 33: 1911-1919Crossref Scopus (73) Google Scholar); however, previous attempts have been unsuccessful to detect and identify the nitrated tyrosine. In this study we present molecular evidence for the specific nitration of bovine PGI2 synthase at tyrosine 430 by high resolution Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry (24Rossier J.S. Youhnovski N. Lion N. Damoc E. Reymond F. Girault H.H. Przybylski M. Angew. Chem. Int. Ed. Engl. 2003; 42: 53-58Crossref Scopus (47) Google Scholar), and the presence of 3-NT upon extended Pronase digestion. We further show an unusually slow digestion by thermolysin, presumably because of a tight fold around the heme, to release a tetrapeptide by an unexpected specific cleavage adjacent to the nitrated tyrosine residue. Pronase from Streptomyces griseus(lyophilized powder) was obtained from Roche Molecular Diagnostics. Thermolysin, type X from Bacillus thermoproteolyticus rokkowas purchased from Sigma. All other chemicals were of analytical grade or highest purity available. PN was a gift from Dr. Koppenol (ETH Zürich, Switzerland) and was synthesized from NO and potassium superoxide according to Kissner et al. (25Kissner R. T. P. Koppenol W.H. Chem. Res. 1997; PubMed Scopus Google Scholar). (CYP a from Bacillus megaterium was a gift from J. A. Peterson and was as S. G. Peterson J.A. J.R. S. C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). against PGI2 synthase was according to et al. Nüsing R. Brugger R. R. R. Ullrich V. FEBS Lett. 1994; PubMed Scopus Google Scholar). against 3-NT was obtained from as a of antibodies and were obtained from The and were purchased from was obtained from and from to bovine were isolated by at in liquid and at were at in a in containing and The was obtained by as V. L. P. Biochem. Pharmacol. 30: PubMed Scopus Google Scholar) to a of with a protein of The additional with PN was with microsomes because active enzyme required for nitration and further the PN at a was by to an protein in were with PN h at The for PGI2 formation of microsomes was by of of of in potassium with for at with the was with of and with of and by was identified by an about The of was with and to of of of and were and by according to the The were for at with and by The proteins were then a by a using a of for The and of proteins was by staining with in in the was with in for h at or at The was then for h with a against PGI2 synthase with the was for with a at a of for and was for of binding according to the to staining with a the was by in under for at and the was with a against 3-NT at a of by a at a of PGI2 synthase were with the PGI2 synthase then or staining with the to for of nitrated of PGI2 synthase from and was by to aortic The was for h at then for h at to a Because by the high of in was by with for h at and of the for at Because with in was by with and for a S. H. Biochem. 1979; PubMed Scopus Google Scholar). for at the and were at the as a white and was by were then in electrophoresis were with for at and then by on staining by was for of C. L. P. Google Scholar). were for in of in with and then for in of The staining was the background white the protein The containing PGI2 synthase was with a in to ions from the gel and several with of PGI2 synthase was by E. A. Biochem. PubMed Scopus Google Scholar). were to in the and with of corresponding to the was then an proteins a were with a were with an to a μm concentrations were with the of of PGI2 synthase were with concentrations of PN and 250 for to and then with to proteases. of Pronase the were for 24 h at then Pronase was and for 24 h at Digestion was with a and for h at to the were with the by for at were on a of a a and and a low 250 from was with a with The was and of were and 3-NT were identified and at and by and The of 3-NT was a 3-NT was with to Because of the of in the protein digestion was than digestion in of PGI2 synthase isolated from μm or were in for at and by on a gel were by staining with as digestion in the gel was according to the of et al. A. F. M. P. A. H. M. S. A. 1996; PubMed Scopus Google Scholar). The protein were from the to and with then with to staining gel and and at in a The was by of the gel and the by the gel were in of containing and 25 thermolysin, and the was digestion was for 24 h at under were several with for 24 h, to and on the above from was with a in with at a of were at and a at were and for further determination of the isolated peptide was by Edman Edman was on an model with an that a at in a phenylthiohydantoin from were under the using in with and in to of were to a and to in a of and were as from the identification of the was by to the and the of a with an at The nitrated tetrapeptide was synthesized on a peptide using peptide synthesis J. A. V. Przybylski M. 2002; PubMed Scopus Google Scholar) with chemicals of analytical grade or highest and other were obtained from the peptide with 3-NT the was with and in of the peptide was with on a resolution mass spectrometry was with a mass with an an ionization and an and a of the of were as K. Przybylski M. Biochem. 2001; PubMed Scopus Google Scholar). The mass were obtained by single of of ionization ionization of was with the and corresponding for mass and were in a of in of mass spectrometry was with a mass with a and a laser and a was with of a of in which was with of the peptide K. Przybylski M. Biochem. 2001; PubMed Scopus Google Scholar). were at an of 25 and were single laser treatment of isolated PGI2 synthase with PN no nitrated could be found of the enzyme with a as well as of the nitrated the presence of nitrated tyrosine. In previous with other P450 proteins a nitration with PN in proteolytic with at from which the of the 3-NT could be identified (13Daiber A. Schöneich C. Schmidt P. Jung C. Ullrich V. J. Inorg. Biochem. 2000; 81: 213-220Crossref PubMed Scopus (35) Google Scholar, 14Daiber A. Herold S. Schöneich C. Namgaladze D. Peterson J.A. Ullrich V. Eur. J. Biochem. 2000; 267: 6729-6739PubMed Google Scholar). Because the M. Daiber A. Herold S. H. Ullrich V. Nitric Oxide. 1999; PubMed Scopus Google Scholar, M.H. Daiber A. Peterson J.A. H. Ullrich V. Biochem. Biophys. 2000; PubMed Scopus Google Scholar) suggested that only active PGI2 synthase can be bovine aortic microsomes were nitrated with increasing concentrations of PN and then the enzyme was isolated by gel electrophoresis Western shown in specific at up to a of 500 μm PN that were by a against PGI2 and a against whereas higher concentrations than 500 μm additional staining of other proteins. The also which was because of the presence of in bovine M.H. M. Ullrich V. Am. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of PN on the of nitration was found up to 250 which was at with the high with the isolated enzyme (16Zou M.H. Ullrich V. FEBS Lett. 1996; 382: 101-104Crossref PubMed Scopus (198) Google Scholar, 17Zou M.H. Martin C. Ullrich V. Biol. Chem. 1997; 378: 707-713Crossref PubMed Scopus (270) Google Scholar), but may be by for PN in the aortic microsomes were to P450 as a model protein for PGI2 synthase its was Because the of PGI2 synthase on the gel low its nitration microsomes were with to not that in microsomes protein for PN and therefore higher PN concentrations are required as with the isolated with other P450 proteins and with model proteins (13Daiber A. Schöneich C. Schmidt P. Jung C. Ullrich V. J. Inorg. Biochem. 2000; 81: 213-220Crossref PubMed Scopus (35) Google A. Herold S. Schöneich C. Namgaladze D. Peterson J.A. Ullrich V. Eur. J. Biochem. 2000; 267: 6729-6739PubMed Google Scholar, K. S. J.S. Biochem. 2001; PubMed Scopus Google Scholar) that Tyr may in this study a PN of 25 μm was that to of a single Tyr residue. The inhibition of was about with the inhibition at 250 the staining at 250 Treatment and of bovine aortic microsomes under provided of PGI2 synthase isolated on from the The protein was and subjected to proteolytic digestion using and mass spectrometric by as well as high resolution C. C. Przybylski M. Biochemistry. 2000; PubMed Scopus Google Scholar) not which yielded peptide identification of the PGI2 synthase no or other peptide were by mass spectrometric low synthase and peptide ions because of additional proteins in low were found by protein not The proteins were to for of the protein cleavage of PGI2 synthase after thermolysin Tyr-containing thermolysin the expected containing tyrosine are of identified Tyr-containing by and from several are for Tyr positions 430 and Tyr-containing were in a the expected containing tyrosine are of identified Tyr-containing by and from several are for Tyr positions 430 and Tyr-containing were The isolated protein was subjected to Pronase digestion under that to release of the 3-NT residue for digestion for h provided Western staining with an but not 3-NT by nitrated as a protein was under digestion for 72 h provided the and of 3-NT after the of about h, suggesting a for in the of the nitration of 3-NT yielded μm 3-NT at a PN of 25 with the of a protein of PGI2 synthase. for of proteins were and a nitration of about could a of than μm with 25 This with the in which also that at higher PN concentrations Tyr may at 25 μm PN only the specific nitration could be The isolated PGI2 synthase was subjected to proteolytic digestion with under a of by mass spectrometric peptide using and These provided the characterization of the structure of the however, of the digestion not in peptide with an at for nitrated tyrosine. with other or also shown not to be on the with Pronase thermolysin was then as a under digestion h, a was found in the protein by at with a of peptide were obtained at suggesting that a of the protein been and was also in the a nitration of PGI2 synthase The peptide was by and Edman which in the structure determination and identification of the nitration site. The mass yielded a single molecular ion corresponding to the of the in a ions were also found of the but not with the precise mass determination of the The of the was ascertained by with thermolysin and of which could for the of the nitrated peptide proof for the nitration at Tyr-430 from Edman which yielded the using 3-NT as a and the mass of the tetrapeptide in the In this study we present the molecular identification of the nitration of PGI2 synthase at tyrosine 430 as an that with the highest for PN. identification of this nitrated as well as Tyr-nitrated was obtained by high resolution in with of in of nitrated by cleavage of the with of NO and hence the ions may the of nitration in proteolytic peptide A. J. Am. 2001; PubMed Scopus Google Scholar). the of the antibodies has been found high to with other it was to the tyrosine residue for the inhibition of enzyme The proteolytic of the Tyr-nitrated was by the high in the at the nitration whereas the in its because of nitrite may and Pronase digestion we could in this study a PN of the 3-NT formation and evidence for the nitration treatment of aortic microsomes with only 25 μm PN a specific nitration at Tyr-430 was established that was important in of the that at higher concentrations a formation of a and may to of other Tyr residues in PGI2 synthase. the for the nitration of PGI2 synthase by PN only about of the enzyme was nitrated as from the staining in the Western blots and about of the to was previous with aortic microsomes (16Zou M.H. Ullrich V. FEBS Lett. 1996; 382: 101-104Crossref PubMed Scopus (198) Google Scholar) we also that the enzyme a of about nitration and that with μm PN about inhibition PN concentrations not in inhibition but from a higher 3-NT formation that Tyr residues other than only 25 μm PN at a of protein are it that only Tyr-430 no other nitrated were of the inhibition of under with of enzyme nitrated to because the gel of isolated not only PGI2 as from other from about purity the μm PGI2 synthase for results provide proof for by PN that has been questioned (9Pfeiffer S. Mayer B. J. Biol. Chem. 1998; 273: 27280-27285Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 10Pfeiffer S. Schmidt K. Mayer B. J. Biol. Chem. 2000; 275: 6346-6352Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 11Pfeiffer S. Lass A. Schmidt K. Mayer B. J. Biol. Chem. 2001; 276: 34051-34058Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 12Pfeiffer S. Lass A. Schmidt K. Mayer B. FASEB J. 2001; 15: 2355-2364Crossref PubMed Scopus (100) Google Scholar). The of this may be provided by the heme involved that the nitration of Tyr residues to the heme (17Zou M.H. Martin C. Ullrich V. Biol. Chem. 1997; 378: 707-713Crossref PubMed Scopus (270) Google Scholar). The low levels of PN from the reaction of NO and O2⨪ under cellular may therefore be for this nitration It be that PGI2 synthase nitration of whereas a nitration of has been found to also occur in a by the of the heme K. Dünstl G. Bachschmid M. Daiber A. Nüsing R. Ullrich V. Nitric Oxide. 2002; 6: 400Google Scholar). The was the low of Tyr-430 that the identification of its nitration for The of of up to at of the expected peptide may be by the high of the protein to digestion. we that Tyr-430 be close to the heme because analogs the because Cys-441 heme at its the Tyr-430 to Cys-441 expected to a around the heme and this structure may be embedded in a This structure the high to and also the for the of the enzyme to and the in Western by on the the of the heme into the protein in systems may be with a tight structure at the active site. and T. of the Tyr-430 residue further to the of this are but by the of the show that a of Tyr-430 by not the suggesting that the may only to the active site. T. and V. This be in with the that nitration could the by than (17Zou M.H. Martin C. Ullrich V. Biol. Chem. 1997; 378: 707-713Crossref PubMed Scopus (270) Google Scholar). results also on the of PN in systems that has been a of in Because of its of than under physiological an for PN and a because the for PN with other present and using isolated PGI2 synthase it was to nitration and inhibition of at levels of PN (16Zou M.H. Ullrich V. FEBS Lett. 1996; 382: 101-104Crossref PubMed Scopus (198) Google Scholar, 17Zou M.H. Martin C. Ullrich V. Biol. Chem. 1997; 378: 707-713Crossref PubMed Scopus (270) Google Scholar), however, with a of present in microsomes higher PN concentrations are This from that show in microsomes no other than in PGI2 synthase up to about 250 by the of PGI2 synthase for nitration was It that PN an efficient for proteins H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and in the presence of metal ions further may for reaction with PN. this the that micromolar PN can PGI2 synthase in endothelial (17Zou M.H. Martin C. Ullrich V. Biol. Chem. 1997; 378: 707-713Crossref PubMed Scopus (270) Google Scholar, M. A. Ullrich V. Rev. 1999; PubMed Scopus (64) Google Scholar) or cells M. A. Ullrich V. Rev. 1999; PubMed Scopus (64) Google Scholar) not to this may be the that PGI2 synthase to the at the (20Spisni E. Griffoni C. Santi S. Riccio M. Marulli R. Bartolini G. Toni M. Ullrich V. Tomasi V. Exp. Cell Res. 2001; 266: 31-43Crossref PubMed Scopus (72) Google Scholar). PN may PGI2 synthase as a the and of the The that endothelial NO synthase found in the compartment that the generation of NO and its reaction with O2⨪ to a and low of may be to PN for the process of we could the nitration of by of aortic and found of and tissue M. S. Zou M.H. Ullrich V. FASEB J. 2003; Google Scholar). of NO synthesis and the presence of superoxide dismutase could the of the The presence of a has been but could not be established K. K. B. K. Martin E. F. F. S. A. 1998; PubMed Scopus Google Scholar, K. F. Biol. Med. 2001; PubMed Scopus Google Scholar). M. and V. may be indeed a of PGI2 synthase that than protein this as a process a be even this the of NO with a of PGI2 by a in of endothelial activation M.H. Jendral M. Ullrich V. Br. J. Pharmacol. 1999; 126: 1283-1292Crossref PubMed Scopus (83) Google Scholar). We Dr. and Damoc for with the and proteolytic digestion We are also to Dr. F. Dr. D. A. of and Dr. C. Schöneich of of for The of Dr. T. Tanabe