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Time-dependent ultraviolet absorption changes in proteins at high pH.

L C Gruen, Z J Tao

    International Journal of Peptide and Protein Research
    |October 1, 1985
    PubMed
    Summary
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    Alkali treatment of proteins causes time-dependent ultraviolet absorption changes, primarily due to modifications in sulfur-containing amino acids like cystine and cysteine. These findings are crucial for accurate protein analysis, especially during tyrosine titration experiments.

    Area of Science:

    • Biochemistry
    • Protein Chemistry
    • Spectroscopy

    Background:

    • Alkali treatment is known to affect protein structure and function.
    • Tyrosine residues in proteins can generate phenolate ions under alkaline conditions, altering UV absorption.
    • The impact of alkali on sulfur-containing amino acids in proteins requires further elucidation.

    Purpose of the Study:

    • To investigate the time-dependent effects of alkali on the ultraviolet absorption of proteins.
    • To identify the specific amino acid residues responsible for these spectral changes.
    • To assess potential interference with tyrosine titration experiments.

    Main Methods:

    • Difference spectrophotometry was employed to monitor UV absorption changes.
    • Proteins were exposed to alkaline conditions in water and 6 M guanidine hydrochloride.

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  • Amino acid analysis data was correlated with spectrophotometric findings.

    • Main Results:

    • Alkali induced time-dependent UV absorption changes beyond those from tyrosine.
    • These changes were more pronounced and rapid in 6 M guanidine hydrochloride.
    • Modifications of cystine and cysteine residues were identified as the primary cause of these spectral shifts.
    • The extent of change correlated with the number and accessibility of sulfur-containing amino acids.

    Conclusions:

    • Alkali-induced degradation of sulfur-containing amino acids contributes significantly to UV absorption changes in proteins.
    • A beta-elimination mechanism is supported, but the reaction pathways are complex.
    • Care must be taken during tyrosine titration experiments to account for non-tyrosine chromophores arising from alkaline degradation.