Camargo, Antonio C. M.

Fink, Edwin

Greene, Lewis J.

Reis, Marina L.

Sampaio, Misako U.

English

Universität München: Elektronischen Publikationen

796
	
Conference Abstracts Vol. 16, No . 5
Aagiotenain (bovine) Asp-Arg-Val-Tyr-Val-Fas-Pro-Phe-IFis-Leu
Gallus Aallux (Angiotenain-F) Aep-Arg-Val-fyr-Val-His-Pro-Phe-Ser-Leu
Elaphe climocophora (Aagiotenain-S) X-Asp-Arg-Val-Tyr-Val-His-Pro-Phe-Tyr-Leu
CHROMATOGRAPHIC SYSTEMS FOR DESALTING AND SEPARATING KININS :
APPLICATION TO TRYPSIN-TREATED HUMAN PLASMA
M.U . Sampaio, M.L . Reis, E. Fink, A.C .M . Camargo and L .J . Greene
Biology Department, Brookhaven National Laboratory, Upton, :Zew York
and
Department of Pharmacology, Faculty of Medicine of Ribeiräo Preto,
University of Sao Paulo, 14100 Ribeirao Preto, ESP, Brasil
An analytical method is presented for the separation of Bradykinin (K-9),
Lys-Bradykinin (R-10), and Met-Lys-Bradykinin (K-11) by equilibrium chroma-
tography on SP-Sephadex, C-25 . The column (0.3 x 50 .0 cm) is operated at room
temperature under equilibrium conditions with 0.02 _M, Tria-HC1 buffer, pH 8 .10,
containing 0 .12 M NaCl . The kinina are identified on the basis of elution
volume and quantitated by bioassay with the isolated guinea pig ileum.
The system was applied to trypain-treated human plasma (1) . The ethanol
soluble fraction was desalted on AG 11-A 8 resin (0.9 x 200 cm) eluted with
H2O and chromatographed on SP-Sephadex at pH 8.1 . Bradykinin was the only
kinin detected in the effluent . However, the bradykinin was not completely
separated from potentiating peptides also released from plasma proteins by
trypain treatment (2) . Bradykinin was separated from the mixture of poten-
tiating peptides by chromatography on SP-Sephadex developed with 0.02 M Tris
HC1 buffer, pft 7 .7, containing 0 .08 _M NaCl . On this basis, plasma-derived
bradykinin potentiating peptides account for 30 to 40 percent of the "apparent"
kinin activity when kinin activity is assayed with the isolated guinea pig
ileum.
The pH 8.1 system has been used to purify commercially available synthetic
K-10 and K-11, which usually contain the lower kinin homologues as well as
oxidized forma of the sulfur in K-11 . Elution profiles of 5-20 micrograms of
synthetic kinins have been obtained by transmission measurements at 206 nm .
These systems can be used to measure the kinina released both as intermediates
and as limit products from kininogen and from plasma by treatment with proteo-
lytic enzymes .
(This research was supported by the United States Atomic Energy Commission,
Fundacäo de Amparo â Pesquisa do Estado de Sâo Paulo and Deutche Forschungs-
gemeinechaft) .
1. C .R . DINIZ and I .F . CARVALHO, Ann . N.Y . Acad . Sci . Z04 77 (1963) .
2 . P .N . AARSEN, Brit . J . Phamiacot . & Chemotherap . 32 453 (1968) .


Chromatographic systems for desalting and separating kinins: Application to trypsin-treated human plasma

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796	Conference Abstracts Vol. 16, No . 5Aagiotenain (bovine) Asp-Arg-Val-Tyr-Val-Fas-Pro-Phe-IFis-LeuGallus Aallux (Angiotenain-F) Aep-Arg-Val-fyr-Val-His-Pro-Phe-Ser-LeuElaphe climocophora (Aagiotenain-S) X-Asp-Arg-Val-Tyr-Val-His-Pro-Phe-Tyr-LeuCHROMATOGRAPHIC SYSTEMS FOR DESALTING AND SEPARATING KININS :APPLICATION TO TRYPSIN-TREATED HUMAN PLASMAM.U . Sampaio, M.L . Reis, E. Fink, A.C .M . Camargo and L .J . GreeneBiology Department, Brookhaven National Laboratory, Upton, :Zew YorkandDepartment of Pharmacology, Faculty of Medicine of Ribeiräo Preto,University of Sao Paulo, 14100 Ribeirao Preto, ESP, BrasilAn analytical method is presented for the separation of Bradykinin (K-9),Lys-Bradykinin (R-10), and Met-Lys-Bradykinin (K-11) by equilibrium chroma-tography on SP-Sephadex, C-25 . The column (0.3 x 50 .0 cm) is operated at roomtemperature under equilibrium conditions with 0.02 _M, Tria-HC1 buffer, pH 8 .10,containing 0 .12 M NaCl . The kinina are identified on the basis of elutionvolume and quantitated by bioassay with the isolated guinea pig ileum.The system was applied to trypain-treated human plasma (1) . The ethanolsoluble fraction was desalted on AG 11-A 8 resin (0.9 x 200 cm) eluted withH2O and chromatographed on SP-Sephadex at pH 8.1 . Bradykinin was the onlykinin detected in the effluent . However, the bradykinin was not completelyseparated from potentiating peptides also released from plasma proteins bytrypain treatment (2) . Bradykinin was separated from the mixture of poten-tiating peptides by chromatography on SP-Sephadex developed with 0.02 M TrisHC1 buffer, pft 7 .7, containing 0 .08 _M NaCl . On this basis, plasma-derivedbradykinin potentiating peptides account for 30 to 40 percent of the "apparent"kinin activity when kinin activity is assayed with the isolated guinea pigileum.The pH 8.1 system has been used to purify commercially available syntheticK-10 and K-11, which usually contain the lower kinin homologues as well asoxidized forma of the sulfur in K-11 . Elution profiles of 5-20 micrograms ofsynthetic kinins have been obtained by transmission measurements at 206 nm .These systems can be used to measure the kinina released both as intermediatesand as limit products from kininogen and from plasma by treatment with proteo-lytic enzymes .(This research was supported by the United States Atomic Energy Commission,Fundacäo de Amparo â Pesquisa do Estado de Sâo Paulo and Deutche Forschungs-gemeinechaft) .1. C .R . DINIZ and I .F . CARVALHO, Ann . N.Y . Acad . Sci . Z04 77 (1963) .2 . P .N . AARSEN, Brit . J . Phamiacot . & Chemotherap . 32 453 (1968) .

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Chromatographic systems for desalting and separating kinins: Application to trypsin-treated human plasma

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