Spectrophotometric Titration of Tyrosine Residues in Human Lysozyme

The ionization of tyrosine residues in human lysozyme was investigated by measurement of spectral changes at pH values between 8.74 and 12.75. Below pH 11.70, at ionic strength 0.10, the titration curve is fully reversible. An average of 4 tyrosine eq per mole are ionized at this pH. At pH values hi...

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Bibliographic Details
Published in:The Journal of biological chemistry 1971-03, Vol.246 (5), p.1457-1460
Main Authors: Latovitzki, N, Halper, J P, Beychok, S
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Chemical Phenomena
Chemistry
Chickens
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Summary:The ionization of tyrosine residues in human lysozyme was investigated by measurement of spectral changes at pH values between 8.74 and 12.75. Below pH 11.70, at ionic strength 0.10, the titration curve is fully reversible. An average of 4 tyrosine eq per mole are ionized at this pH. At pH values higher than 11.70, the spectra are time-dependent. Reversal from any pH above 11.7, after 12 to 30 hours, leads to precipitation. Analysis of the results suggests that of the 6 tyrosine residues in the molecule, 3 are readily accessible and titrate as equivalent groups with pK int = 9.81. A 4th residue is inaccessible and can be titrated only after unfolding. The remaining 2 titrate with anomalously high pK values. When, on the average, more than one of these residues are ionized, a time-dependent exposure of the buried tyrosine residue occurs. A comparison of these results with the ionization behavior of the 3 tyrosine residues in hen egg white lysozyme is presented and discussed in terms of differences in the sequences of the two enzymes.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)76993-2