Exploring the protease inhibitory potential of 2-bromo-2-nitro-1,3- propanediol (Bronopol): Initial findings.

The compound 2-bromo-2-nitropropane-1,3-diol also known as Bronopol (BP) is a widely used antimicrobial preservative which highly soluble in water. Although its antimicrobial mechanism has been well described, its potential to modulate protease activity has not been previously examined. Proteases represent one of the largest and most industrially important classes of enzymes, and protease inhibitors are essential tools in research, biotechnology, and therapeutic development. In this study, we investigated the ability of BP to inhibit serine, cysteine, and aspartic proteases using both synthetic and natural proteins. Protease inhibition was quantified using trypsin, proteinase K, papain, and pepsin. BP produced a clear dose-dependent inhibition of all proteases tested, with distinct sensitivity profiles. Proteinase K and trypsin activities declined progressively across 50-1000 μM BP, reaching approximately 20% residual activity at the highest concentration. Papain showed the greatest susceptibility, exhibiting near-complete inhibition (>95%) at 500-1000 μM. Pepsin displayed modest inhibition at low BP concentrations but substantial loss of activity above 250 μM. Proteolytic cleavage of bovine serum albumin (BSA) and ovalbumin were assessed by SDS-PAGE. Proteinase K alone caused extensive degradation of both substrates; however, BP at 2500-10 000 μM markedly reduced proteolysis and preserved full-length proteins, confirming protective effects observed in enzymatic assays. Together, these findings identify BP as an inhibitor of serine, cysteine, and aspartic proteases, with potent effects at moderate to high concentrations. This previously unreported activity suggests additional functional roles for BP in biochemical systems and highlights its potential relevance in research and industry.