Second-Derivative Spectroscopy of Proteins

A Method for the Quantitiative Determination of Aromatic Amino Acids in Proteins

Authors

  • Ciro BALESTRIERI,

    1. Istiuto di Chimica Biologica 1a Facoltè di medicina e Chirurgia, Universitè-degli Studi, Via Costantinopoli 16, 1-801138 Napoli, Italy
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  • Giovanni COLONNA,

    1. Istiuto di Chimica Biologica 1a Facoltè di medicina e Chirurgia, Universitè-degli Studi, Via Costantinopoli 16, 1-801138 Napoli, Italy
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  • Alfonoso GIOVANE,

    1. Istiuto di Chimica Biologica 1a Facoltè di medicina e Chirurgia, Universitè-degli Studi, Via Costantinopoli 16, 1-801138 Napoli, Italy
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  • Gaetano IRACE,

    1. Istiuto di Chimica Biologica 1a Facoltè di medicina e Chirurgia, Universitè-degli Studi, Via Costantinopoli 16, 1-801138 Napoli, Italy
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  • Luigi SERVILLO

    1. Istiuto di Chimica Biologica 1a Facoltè di medicina e Chirurgia, Universitè-degli Studi, Via Costantinopoli 16, 1-801138 Napoli, Italy
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Abstract

Second derivative spectroscopy has been used to resolve the complex protein spectrum in the near-ultraviolet region and the contributions of the three aromatic chromophores have been evaluated.

A method for the direct quantitative determination of phenylalanine and tryptophan in proteins has been carried out. Phenylalanine determination has been carried out in the spectral region between 250 and 265 nm, where there are no significant contributions from other aromatic chromophores. Tryptophan determination has been performed in the 290–295-nm region and the experimental values have been corrected for the presence of tyrosine.

The results obtained on 10 highly purified proteins have been found in good agreement with those obtained from sequence analysis.

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