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Study of isotope effects on protein binding by gas chromatography/mass spectrometry of theophylline–phenobarbitone and 2 H, 13 C, 15 N isotopomers
Author(s) -
Cherrah Y.,
Falconnet J. B.,
Desage M.,
Brazier J. L.,
Zini R.,
Tillement J. P.
Publication year - 1988
Publication title -
biomedical and environmental mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 0887-6134
DOI - 10.1002/bms.1200170403
Subject(s) - theophylline , chemistry , human serum albumin , isotopomers , mass spectrometry , chromatography , tryptophan , isotope , kinetic isotope effect , organic chemistry , biochemistry , amino acid , molecule , deuterium , pharmacology , medicine , physics , quantum mechanics
Abstract We describe a comparative study of human serum albumin (HSA) binding by equilibrium dialysis (pH 7.4, 37°C, 3 h) for two groups of isotopic analogues: theophylline and 1‐C( 2 H 3 )theophylline; unlabelled, 5(ethyl( 2 H 5 )),‐5(phenyl( 2 H 5 )) and 1,3‐ 15 N;2‐ 13 C‐phenobarbitone. Bound and free drug fractions are quantified by combined gas chromatography/mass spectrometry. In three instances, protein binding parameters are greatly affected by isotopic substitution, namely for: theophylline and 1‐C( 2 H 3 )theophylline with isotope effects on total binding site concentration ( N ), affinity constant ( K a ) and extent of HSA binding (%) respectively, equal to: N L /N H =0.51; K aL /K aH = 1.78; %L/%H = 0.96 (L (light) and H (heavy) represent the unlabelled and labelled analogue respectively); phenobarbitone/‐5‐(phenyl( 2 H 5 ))phenobarbitone, N L /N H = 1.72; K aL / K aH = 0/56; %L/%H = 1.26; phenobarbitone/1,3‐ 15 N;2‐ 13 C phenobarbitone, N L /N H = 2.95; K aL / K aH = 0.44; %L/%H = 1.32, together with a change from one (saturable) to two (saturable + non‐saturable) families of albumin binding sites in the latter case. Contrasting with these data, no HSA binding isotope effect was observed on phenobarbitone C5 ethyl deuteration.