Urease from Helicobacter pylori is inactivated by sulforaphane and other isothiocyanates

Abstract

Infections by Helicobacter pylori are very common, causing gastroduodenal inflammation including peptic ulcers, and increasing the risk of gastric neoplasia. The isothiocyanate (ITC) sulforaphane [SF; 1-isothiocyanato-4-(methylsulfinyl)butane] derived from edible crucifers such as broccoli is potently bactericidal against Helicobacter, including antibiotic-resistant strains, suggesting a possible dietary therapy. Gastric H. pylori infections express high urease activity which generates ammonia, neutralizes gastric acidity, and promotes inflammation. The finding that SF inhibits (inactivates) urease (jack bean and Helicobacter) raised the issue of whether these properties might be functionally related. The rates of inactivation of urease activity depend on enzyme and SF concentrations and show first order kinetics. Treatment with SF results in time-dependent increases in the ultraviolet absorption of partially purified Helicobacter urease in the 260-320 nm region. This provides direct spectroscopic evidence for the formation of dithiocarbamates between the ITC group of SF and cysteine thiols of urease. The potencies of inactivation of Helicobacter urease by isothiocyanates structurally related to SF were surprisingly variable. Natural isothiocyanates closely related to SF, previously shown to be bactericidal (berteroin, hirsutin, phenethyl isothiocyanate, alyssin, and erucin), did not inactivate urease activity. Furthermore, SF is bactericidal against both urease positive and negative H. pylori strains. In contrast, some isothiocyanates such as benzoyl-ITC, are very potent urease inactivators, but are not bactericidal. The bactericidal effects of SF and other ITC against Helicobacter are therefore not obligatorily linked to urease inactivation, but may reduce the inflammatory component of Helicobacter infections.

Figure 1

Microtiter well calibration curve for measurement of ammonia by change in absorption of phenol red. Aliquots of NH₄OH were added to 200 μL of 100 mM potassium phosphate, pH 6.8, containing 0.002% phenol red, and the changes in absorption at 570 nm (○) and the pH (●) determined. The insets show the absorption spectra of phenol red upon addition of increasing quantities of NaOH, and the structure of the chromophore.

Figure 2

Inactivation of ureases by a series of concentrations of sulforaphane (A,B,C) and boric acid (D). The ureases from (A) H. pylori strain J99 and (B) jack bean were incubated for 1 h with inactivator prior to assay. The ureases from (C) 4 strains of H. pylori (60190, 26695, SS1, J99) and jack bean (JB), were incubated for 2 h with sulforaphane (SF) prior to assay. (D) Urease from H. pylori strain J99 was incubated with boric acid (BA) for 1 h prior to assay. Urease activities were measured in the presence of urea 120 (○), 100 (△), 80 (▽), 60 (◇), 40 (□), and 20 (+) mM urea.

Figure 3

Time-course of inactivation of partially purified H. pylori urease by a series of concentrations of sulforaphane (SF). The urease concentrations were 26.7 μg/mL (A) and 80 μg/mL (B), and the remaining urease activities were measured in the presence of 20 mM urea. Semi-log plots of residual activity with respect to time of inactivation are linear, consistent with a first-order inactivation process.

Figure 4

(A) Time-course of difference spectra of partially purified H. pylori urease (9 μg/mL) treated with 740 μM SF for 12, 30, 76, and 120 min. The control cuvette contained the equivalent concentration of sulforaphane. (B) Change in absorption at 280 nm in 60 min when sulforaphane (7.4 – 740 μM) was added to partially purified H. pylori urease (30 μg/mL).