Purificação e caracterização parcial de tonina símile a partir de placenta de humano

Abstract

The existence of a renin-angiotensin system (RAS) in the female reproductive organ has been demonstrated. In this tissue, RAS could be acting independently or in accord with the circulating system, participating in the regulation of some functional aspects, in situations of abnormalities also evidenced during the gestation. All the components of RAS, including AT1 receptor had been identified in human placenta. Tonin is one serineproteinase present in many rat tissues that is able to cleave angiotensin II (Ang II) from angiotensin I (Ang I) and directly from angiotensinogen. This work was developed with the objective to isolate and to characterize enzyme(s) with tonin simile activity from human placenta. Placenta from health women had been gotten in public hospital, after formal assent of the patient. The placenta was placed in dry ice for the transport and later stored at -80°C. For processing, the placenta was defrosted and washed with physiological solution in abundance for withdrawal of the blood excess. Placenta fragments were cut and homogenized in 0,25 M sucrose pH 7,0 containing enzymatic inhibitors. The crude homogenate was treated with ammonium sulphate to 25, 50 and 80% for precipitation of proteins. Each precipitated was resuspended in water, dialysed and the Ang II liberating activity measured by radioimmunoassay after incubation of the fractions with partially purified human angiotensinogen and the tetradecapeptide synthetic renin substrate (AG(1-14)). The Ang II liberating activity was found in the 50% ammonium sulphate fraction. Aliquots of this fraction were load in chromatography in Superdex HR-200 column. The active fractions from Superdex HR-200 column had been concentrated against polyethylene glycol 6000, submitted to dialysis against Tris-HCl 20 mM pH 8.4 buffer and load in Q-Sepharose column that was eluted with Tris buffer in an initial stage followed by NaCl gradient. The active fraction that eluted with 100 mM NaCl was load in Phenyl- Superose column. This column was eluted with a linear gradient (1.7 to 0.0 M) of ammonium sulphate. The Ang II liberating activity was determined at each stage of purification. In addition, to each stage, the active fractions were submitted to polyacrylamide gel electrophoresis for identification of the molecular mass of the active component. Tests of inhibition with 10 mM PMSF, 1 and 10 mM pepstatin and 1 mM aprotinin had been carried out for active fractions eluted from Q-Sepharose. The presence of angiotensinase activity also was searched in these fractions, as well as the Ang I liberating activity. Our results show the presence of two Ang II liberating fractions from human placenta, both inhibited by PMSF. This activity was not inhibited by pepstatin or by aprotinin. It was not detected angiotensinase activity and Ang I liberating activity on the active fractions. The molecular mass estimated for the active fractions is around 52 kDa and the N-terminal sequence found for the protein was EVQLVESGGGL.