[Fluorescence spectra analysis of papain treated by pulsed electric field].

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 x cm(-1), frequency 1500 Hz, pulse width 40 micros 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 alpha-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 alpha-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.

AI evidence extraction

Main findings

Papain relative activity decreased by 56.5% after pulsed electric field treatment (50 kV/cm, 1500 Hz, 40 micros pulse width, 19,800 pulses). Treated samples showed higher fluorescence excitation intensity (at 280 nm: 80 untreated vs 120 treated), an emission peak shift from 342 nm to ~346 nm, and lower fluorescence polarization, interpreted as loosening/breakdown of alpha-helix structure and exposure/release of fluorescent amino acid residues, contributing to enzyme inactivation.

Outcomes measured

• Papain relative enzymatic activity
• Fluorescence excitation spectrum
• Fluorescence emission spectrum (peak shift)
• Fluorescence polarization degree
• Protein secondary structure (alpha-helix loosening/breakdown inferred from spectra)

Limitations

• No sample size reported in abstract
• In vitro enzyme study; relevance to human/animal health not addressed
• Only one set of exposure conditions explicitly reported in abstract

{
    "study_type": "in_vitro",
    "exposure": {
        "band": null,
        "source": "pulsed electric field (PEF)",
        "frequency_mhz": null,
        "sar_wkg": null,
        "duration": "pulse width 40 micros; pulse number 19,800; frequency 1500 Hz"
    },
    "population": null,
    "sample_size": null,
    "outcomes": [
        "Papain relative enzymatic activity",
        "Fluorescence excitation spectrum",
        "Fluorescence emission spectrum (peak shift)",
        "Fluorescence polarization degree",
        "Protein secondary structure (alpha-helix loosening/breakdown inferred from spectra)"
    ],
    "main_findings": "Papain relative activity decreased by 56.5% after pulsed electric field treatment (50 kV/cm, 1500 Hz, 40 micros pulse width, 19,800 pulses). Treated samples showed higher fluorescence excitation intensity (at 280 nm: 80 untreated vs 120 treated), an emission peak shift from 342 nm to ~346 nm, and lower fluorescence polarization, interpreted as loosening/breakdown of alpha-helix structure and exposure/release of fluorescent amino acid residues, contributing to enzyme inactivation.",
    "effect_direction": "harm",
    "limitations": [
        "No sample size reported in abstract",
        "In vitro enzyme study; relevance to human/animal health not addressed",
        "Only one set of exposure conditions explicitly reported in abstract"
    ],
    "evidence_strength": "low",
    "confidence": 0.7399999999999999911182158029987476766109466552734375,
    "peer_reviewed_likely": "yes",
    "keywords": [
        "papain",
        "pulsed electric field",
        "PEF",
        "fluorescence spectra",
        "tryptophan",
        "tyrosine",
        "phenylalanine",
        "enzyme activity",
        "protein structure",
        "alpha-helix"
    ],
    "suggested_hubs": []
}

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