Abstract：Tryptophane, tyrosine and phenylalanine are there kinds of fluorescent amino acids and exist in papain, so the structure change of papain can be measured by fluorescence spectra analysis without exterior fluorescence probe. Fluorescence excitation spectrum, emission spectrum and polarization spectrum were used to analyze the possible mechanisms of papain’s activity change after being treated by pulsed electric field (PEF). Results demonstrated that the relative activity of papain was decreased by 56.5％ after PEF treatment under the condition of electric field strength 50 kV·cm-１,frequency 1 500 Hz, pulse width 40 μs and pulse number 19 800. The spectra of fluorescence excitation showed that the relative fluorescence strength of the treated sample was distinctly higher than the untreated one, even at peak position (280 nm) which was 80 and 120 for untreated and treated samples, respectively. The peak position in the fluorescence emission spectrum of treated sample was shifted from the original 342 nm to about 346 nm, and its fluorescence polarization degree was much smaller compared to the untreated sample. These phenomena indicated that the α-helix structure of papain was loosened or broken down after being treated by PEF. This treatment made the amino acid’s residue exposed from inside to outside, and even some fluorescent amino acids such as tryptophane, tyrosine and phenylalanine were decomposed from the α-helix structure of protein and went into the solution which helped increase the fluorescent strength. This effect led to the active site change of the enzyme and finally inactivated it.
Key words：Pulsed electric field;Papain;Activity of enzyme;Fluorescence spectrum
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