Harold S. Olcott

Isolation and characterization of a trypsin inhibitor from lima beans

Abstract 1. 1. The purification of a lima bean protein fraction of high trypsin-inhibiting activity is described. The active protein was freed from inert crystallizable protein. 2. 2. The inhibitor shows remarkably high sulfur (5.5%) and cystine (16%) contents. Preliminary amino acid analyses are presented. 3. 3. The inhibitor is relatively resistant to denaturing conditions. It is not attacked by pepsin or papain. 4. 4. The average molecular weight of the concentrate was approximately 10,000 by osmotic pressure and ultracentrifugal analysis. 5. 5. The inhibitor is not inactivated by extensive esterification of its carboxyl groups, or by iodination and coupling of its phenolic and imidazole groups. Blocking of part of the amino and amide or guanidyl groups, sulfation of the hydroxyl groups, or extensive reduction of the disulfide bonds destroys its activity. 6. 6. Marked differences exist between the properties and essentiality of groups of the lima bean inhibitor and those of ovomucoid and the soybean inhibitor.

Chemical Derivatives of Gramicidin

Summary Gramicidin was treated with a number of reagents which were known, or could be shown to combine with, either its indole, or its hydroxyl groups, or both. The products were prepared and assayed in search of a water-soluble derivative of low hemolytic and high antibacterial activity. Formaldehyde had previously been shown to decrease selectively the hemolytic activity, but to render gramicidin only slightly less water-insoluble. 1. A glyoxylic acid derivative was obtained, the sodium salt of which was water-soluble. The product, however, retained most of the original hemolytic activity, and attempts to find experimental conditions under which the advantages of the glyoxylic and formaldehyde treatment could be combined were not successful. 2. An indole sulfamate of gramicidin was obtained with pyridine-chlorosulfonic acid, the salts of which were readily soluble in aqueous alcohol. The product showed somewhat greater decreases in hemolytic, than in antibacterial activity. 3. The succinic half esters of gramicidin and methylol gramicidin showed the greatest promise. The succinyl derivative of gramicidin, containing one succinyl group for each ethanolamine residue, gave a water-soluble sodium salt. Its hemolytic activity was slightly decreased and could be further lowered by subsequent formaldehyde treatment. Notwithstanding the in vitro hemolytic activity, the derivative appeared to be remarkably nontoxic by intravenous injection. The succinic anhydride derivative of methylol-gramicidin, containing one succinyl for each indole-methylol plus each original hydroxyl group, was lowered in all, but markedly more in hemolytic and toxic than in antibacterial activities. Its sodium salt was water-soluble even in the presence of other ions, but not very stable at neutrality.