Measurement of β-Lactamase Activity and Rate of Inactivation of Penicillins by a pH-Stat Alkalimetric Titration Method

The pH-stat alkalimetric titration method was shown to be applicable and superior to other chemical methods in the investigation of β-lactamase activity as well as in kinetic studies. For both staphylococcal and bacillary β-lactamases, the pH optimums depended not only on bacterial strain and temperature but also on the substrate. Of all the compounds studied, 6-[2-amino-(3-trienyl)acetamido]penicillanic acid, 6-[2-(1-aminocyclohexyl)acetamido]penicillanic acid, 6-(2-amino-2-methylpropionamido)penicillanic acid, and ampicillin were the least stable toward Staphylococcus aureus Tex-2 β-lactamase. Penicillin G, phenoxymethyl penicillin, phenethicillin, 6-(1-amino-3-cyclopentene-1-carboxamido)-penicillanic acid, 6-(2-amino-2-indancarboxamido)penicillanic acid, and carbenicillin were about twice as stable as ampicillin, and 6-(1-aminocyclopentanecarboxamido)penicillanic acid, 6-[2-(1,4-cyclohexadien-1-yl)acetamido]penicillanic acid, and cyclacillin were about four times as stable. N-Sulfonylation of cyclacillin gave a product, 6-[1-(2-mesitylenesulfonamido)cyclohexanecarboxamido]-penicillanic acid, that was about 10 times as stable as the parent compound. The nature of the penicillin side chain, particularly the stereospecificity, plays a significant role in the stability of this molecule toward the β-lactamases.