Fluorometric analysis of native, urea-denatured and refolded human prostatic acid phosphatase.
Human prostatic acid phosphatase (EC 3.1.3.2) was denatured in 6 M urea at pH 2.5, but was refolded by dilution at pH 7.0, as demonstrated by the recovery of nearly complete enzyme activity and dimeric structure. The conformational changes among the native, denatured and refolded states were monitored by means of steady-state and nanosecond pulse fluorometry of tryptophan residues of the enzyme. The relative quantum yield of the fluorescence was highest in the native enzyme and lowest in the denatured one, and was intermediate in the refolded enzyme, although the emission peak was reproducible after refolding. The observed decay curves of tryptophan fluorescence of the native, denatured and refolded states were analyzed by decay functions of three lifetimes. The fluorescence lifetimes of the refolded enzyme were shorter than those of the native one. The fluorescence of the denatured enzyme decayed much faster than that of the other forms. The fluorescence excitation spectra revealed that the excitation energy of phenylalanine was transferred to tryptophan(s) in the native and refolded forms, but not in the denatured form. The efficiency of the energy transfer was higher in the native enzyme than in the refolded one. It was found by excitation polarization spectra that the freedom of internal motion of tryptophans was greater in the refolded enzyme than in the native enzyme. In the denatured enzyme the polarization anisotropies were very low. These results indicate that the higher structure with respect to tryptophans of the refolded enzyme is delicately but definitely different from that of the native enzyme and that local conformation of the active center is recovered upon refolding.
Ostrowski
W S
WS
Institute of Medical Biochemistry, N. Copernicus Academy of Medicine, Krakòw, Poland.
Kuciel
R
R
Tanaka
F
F
Yagi
K
K
D000135:Acid Phosphatase / Q000737:chemistry*; D005454:Fluorescence Polarization; D006801:Humans; D008297:Male; D011467:Prostate / Q000201:enzymology*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis
D014364:Tryptophan; D000135:Acid Phosphatase