Nonaromatic amino acids in the combining site region of a monoclonal anti-spin-label antibody

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Abstract
The NMR spectra of monoclonal Fab antibody fragments were recorded in the absence and presence of the specific spin-label dinitrophenyl hapten. The difference spectra reveal the presence of about 50 amino acids in the region of the combining site. By selective deuteration and by use of double difference spectra, all the resonances in the spectral region -1 to 1.5 ppm were identified. In the combining site region there are 4 or 5 valines, certainly 3 and possibly 5 threonines, 3 or 4 leucines, 2 or 3 isoleucines, and 6 or 7 alanines. Selective deuteration of methionine and lysine reveals 1 methionine and 2 lysines in the difference spectra. All of these amino acids are estimated to be within 17 .ANG. of the paramagnetic hapten. By using difference spectra involving low fractional occupancy of the combining site with the spin-label hepaten, it is established that 1 threonine and 1 valine are very close to the paramagnetic hapten.

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