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 angiotensin-converting enzyme (ACE), for the determination of its tryptophan and tyrosine contents and to follow-up guanidine denaturation. ACE purity was assessed by both SDS-PAGE and capillary electrophoresis performed 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 x 10(5) (mol/l)-1 x cm-1. Denaturation of ACE by 6 mol/l guanidine hydrochloride produced a hypochromic effect of 23% at 280 nm and led to a blue shift of 3.5 nm. In guanidine solution, absorbance measurements at 288 and 280 nm predicted a ratio of 1 between tyrosine and 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 spectrum, in parallel with the complete loss of enzymatic activity although the protein remained whole as judged on SDS-PAGE. We also re-evaluated ACE zinc content by atomic absorption spectroscopy and demonstrated that ACE molecule obviously contains two zinc atoms.
Baudin
B
B
Hôpital Saint-Antoine, Paris, France.
Mario
N
N
Gaba
S
S
Giboudeau
J
J
D000818:Animals; D004229:Dithiothreitol / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008168:Lung / Q000201:enzymology*; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011489:Protein Denaturation; D013054:Spectrophotometry, Atomic / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013439:Sulfhydryl Reagents / Q000737:chemistry; D013552:Swine; D014364:Tryptophan / Q000032:analysis; D014443:Tyrosine / Q000032:analysis; D015032:Zinc / Q000032:analysis
D006146:Guanidines; D013439:Sulfhydryl Reagents; D014443:Tyrosine; D014364:Tryptophan; D007703:Peptidyl-Dipeptidase A; D015032:Zinc; D019791:Guanidine; D004229:Dithiothreitol