Identification of methyl resonances in the 1H NMR spectrum of incubated blood cell lysates

The origin of low frequency methyl resonances which appear in the spin-echo 1H nuclear magnetic resonance spectra of incubated blood cell lysates was investigated by several techniques including 1H and 13C nuclear magnetic resonance spectroscopy, electrophoresis, high performance liquid chromatography, gel filtration, and amino acid analysis. These resonances were identified as arising from methyl moieties of leucine and valine. Other peaks which also appeared in the spectra of incubated blood cell lysates were assigned to methyl groups of alanine and threonine. The free amino acids are products of neutral proteases located on the leukocyte membrane or able to act on the extracellular medium. Since more than one enzyme appears to be implicated, it is possible that both membrane and granule proteases take part in the hydrolysis. Comparison of rates of product formation in white cell lysates incubated with human serum albumin, and with red cell lysate, suggests that erythrocyte peptidases also contribute to proteolysis in the latter case.