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Solution composition dependent variation in extinction coefficients for p-nitroaniline.

R Lottenberg, C M Jackson

    Biochimica Et Biophysica Acta
    |February 15, 1983
    PubMed
    Summary
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    Para-nitroaniline extinction coefficients change with solution composition, affecting measurements. Using a difference extinction coefficient at 381 nm minimizes errors caused by these spectral shifts.

    Area of Science:

    • Analytical Chemistry
    • Spectroscopy
    • Biochemistry

    Background:

    • Extinction coefficients of para-nitroaniline (p-NA) are crucial for spectrophotometric quantification.
    • Solution composition can influence spectral properties, leading to potential measurement errors.

    Purpose of the Study:

    • To investigate the impact of solution composition on p-nitroaniline extinction coefficients.
    • To identify strategies for mitigating spectral shifts in p-nitroaniline measurements.

    Main Methods:

    • Spectrophotometric analysis of p-nitroaniline in solutions of varying ionic strength.
    • Evaluation of spectral shifts induced by additives like poly(ethylene glycol) and bovine albumin.
    • Determination of isosbestic wavelengths for peptide p-nitroanilide/p-nitroaniline mixtures.

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    Main Results:

    • The absorption spectrum of p-nitroaniline exhibits a red-shift with increasing ionic strength.
    • Additives such as poly(ethylene glycol) and bovine albumin also cause spectral shifts.
    • Isosbestic wavelengths in peptide p-nitroanilide/p-nitroaniline mixtures are sensitive to solution composition.

    Conclusions:

    • Solution composition significantly affects p-nitroaniline extinction coefficients, necessitating careful consideration in experimental design.
    • A difference extinction coefficient at 381 nm (p-nitroaniline's maximum wavelength) is proposed to reduce errors from spectral shifts.
    • This approach enhances the reliability of spectrophotometric assays involving p-nitroaniline.