SPECTROSCOPIC STUDIES ON ANGIOTENSIN I-CONVERTING ENZYME

We used near and far UV spectrophotometry for the re-evaluation of the molar extinction coefficient at 280 nm (epsilon(280)) Of pulmonary an giotensin-converting enzyme (ACE), for the determination of its trypto phan and tyrosine contents and to follow-up guanidine denaturation. AC E purity was assessed by both SDS-PAGE and capillary electrophoresis p erformed in denaturing conditions. The maxima of the near UV spectrum of purified ACE, dissolved in phosphate buffer pH 6.5, was at 279 nm; with an estimated M(r) of 160 kD, epsilon(280) of native ACE was 1.5 /- 0.05 . 10(5) (mol/l)(-1). cm(-1). Denaturation of ACE by 6 mol/l gu anidine hydrochloride produced a hypochromic effect of 23% at 280 nm a nd led to a blue shift of 3.5 nm. In guanidine solution, absorbance me asurements at 288 and 280 nm predicted a ratio of 1 between tyrosine a nd tryptophan, whereas it was 1.8 with the measure of the amplitude of the spectral bands at 283 and 292 nm of the second derivative of the near UV spectrum. Unfolding of the peptide chain in 6 mol/l guanidine was also well characterized by the second derivative of the far UV spe ctrum, in parallel with the complete loss of enzymatic activity althou gh the protein remained whole as judged on SDS-PAGE. We also re-evalua ted ACE zinc content by atomic absorption spectroscopy and demonstrate d that ACE molecule obviously contains two zinc atoms.