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Purpose of further explore rat dipeptidyl peptidase Ⅰ (Tripeptidyl peptidase I, TPP I) the biological activity of, particularly small peptides having the biological activity of the degradation mechanism, using high performance liquid chromatography and flight mass spectrometer, and in vitro analysis of the rat TPP Ⅰ hydrolysis of the small peptides of the angiotensin I, II, III, etc.. Method first purified rat TPP I and angiotensin I, II, III Leu--enkepha, LIN, bradykinin and other small peptides and synthetic substrate Ala-Ala-Phe-MAC mixing, 37 ℃ water bath at different hydrolysis times. observe TPP I hydrolysis of these small peptides. The hydrolysis mixture after digestion with a 0.22 μm membrane by suction, respectively, after taking the filtrate the 50μl sample addition to the reverse-phase high-performance liquid chromatography column, renin - angiotensin I, II with 0.01% TFA / acetonitrile concentration successive increments from 0-50% elution; renin - angiotensin III, Leu-enkephalin, Bradykinin 0.01% TFA / acetonitrile concentration successive increments from 0-100% elution in the A215nm wavelength collect each peak. Flight mass spectrometer detection board, each detection drip plus of 0.5μl Matrix and 2μl of each peak collected liquid, mix dry naturally. Completely dry samples of all to be seized on the machine. To obtain TPPI cutting angiotensin I, II, III, and Leu-enkephalin, Bradykinin respective molecular weight of the peptides map, using a fluorescence spectrophotometer in Ex: 380nm, em: 440nm conditions measured under the synthetic substrate Ala-Ala- Phe-MCA fluorescence intensity, and the release of the AMC standard curve 1 TPPi Ang I hydrolysis occurs in DRV ↓ YIHPFHL, release tripeptide DRV, and heptapeptide YIHPEHL two hydrolysis fragments. The TPPI hydrolysis occurs in Ang II the DRV and YIHPF between release tripeptide DRV and pentapeptide YIHPF, two hydrolysis fragments. The TPPI occurred on the hydrolysis of Ang III between RVY and IHPF release the tripeptide RVY and tetrapeptide IHPF two hydrolysis fragment-line method to calculate the rate of hydrolysis of the enzyme to the substrate. Phase results through experiment proved, Ang I, II, and III of the content is gradually reduced with the enzymatic time prolonged. 16h when the rate of hydrolysis of the Ang I was 11.3%; 16h Ang II of the hydrolysis rate of 18.2%; 15min of Ang III a hydrolysis rate of 95%, 30min, Ang III disappeared completely. 24h after Leu-enkephalin, Bradykinin still no hydrolysis signs. 2, using the concentration of the AMC to the fluorescence intensity of the standard curve, Find of TPP I on the size of the rate of hydrolysis of the Ala-Ala-Phe-MCA, indicating that at 60 min, TPP I, the rate of hydrolysis of the Ala-Ala-Phe-MCA 33.8%. Conclusion 1, TPP I can to varying degrees hydrolysis of Ang I, II, III, no hydrolysis of Leu-enkephalin, Bradykinin. The TPP I Ang III The hydrolysis of the most powerful, and far greater than the speed of the hydrolysis of Ala-Ala-Phe-MCA. That the rate of hydrolysis of the order: Ang III gt; gt; Ala-Ala-Phe-MCA> Ang II gt; Ang I. 2, angiotensin III TPP I natural matrix.
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