Reactivities of neutral and cationic forms of 2,2′-dipyridyl disulphide towards thiolate anions. Detection of differences between the active centres of actinidin, papain and ficin by a three-protonic-state reactivity probe

Abstract

The second-order rate constants (k) for the reactions of 2,2'-dipyridyl disulphide (pKa2,45) with 2-mercaptoethanol (pKa9.6) and with benzimidazol-2-ylmethanethiol (pKa values 5.6 and 8.3) were determined at 25 degrees C at I 0.1 by stopped-flow spectral analysis over a wide range of pH. These were used to calculate the pH-independent second-order rate constants (k) for the reactions of neutral 2,2'-dipyridyl disulphide and of its monocation with the 2-mercaptoethanol thiolate anion (associated pKa9.6) and with the benzimidazol-2-ylmethanethiol zwitterion (associated pKa5.6). For both thiolate ions, the rate-enhancement factor (kmonocation/kneutral disulphide) is about 1.5x10(3). The dependence on pH in acidic media of k for the reaction of 2,2'-dipyridyl disulphide with actinidin, the thiol proteinase from Actinidia chinensis, was shown to differ from the forms of pH-dependence observed for the analogous reactions with papain (EC 3.4.22.2) and ficin (3.4.22.3). The reactivity of the 2,2'-dipyridyl disulphide dication and its apparent sensitivity to the presence and location of a positive charge in the attacking thiol are discussed.

Cryotolerance of stallion spermatozoa is related toROSproduction and mitochondrial membrane potential rather than to the integrity of sperm nucleus

“…The levels of free thiol radicals were determined in all stallion sperm samples by using the 2,2′‐dithiodipyridine technique (2,2′‐dipyridyl disulphide; Sigma, Saint Louis, MO, USA) as described in Brocklehurst et al . (). With this purpose, 10 μL of the prepared sperm samples obtained as described above were added to 990 μL of an aqueous solution of 0.4 m m 2,2′‐dithiodipyridine, prior to incubation at 37.5 °C for 1 h. Levels of free thiol groups in sperm head proteins were determined via spectrophotometric analysis at a wavelength of 343 nm, per triplicate.…”

Melatonin affects the motility and adhesiveness of in vitro capacitated boar spermatozoa via a mechanism that does not depend on intracellular ROS levels

“…supernatants and Tris-solubilised pellets) were determined using the 2,2 0 -dithiodipyridine technique (2,2 0 -dipyridyl disulphide; Sigma-Aldrich) as described in Brocklehurst et al (1979). supernatants and Tris-solubilised pellets) were determined using the 2,2 0 -dithiodipyridine technique (2,2 0 -dipyridyl disulphide; Sigma-Aldrich) as described in Brocklehurst et al (1979).…”

“…Levels of free cysteine radicals in both fractions (i.e. supernatants and Tris-solubilised pellets) were determined using the 2,2 0 -dithiodipyridine technique (2,2 0 -dipyridyl disulphide; Sigma-Aldrich) as described in Brocklehurst et al (1979). Briefly, 10 lL of the supernatant or resuspended pellet was added with 990 mL of an aqueous solution containing 0.4 mM 2,2 0 -dithiodipyridine.…”

The achievement of boar spermin vitrocapacitation is related to an increase of disrupted disulphide bonds and intracellular reactive oxygen species levels

“…Levels of free cysteine residues in both tail and head fractions were determined with 2,2 0 -dithiodipyridine technique (2,2 0dipyridyl disulphide; Sigma-Aldrich) as described by Brocklehurst et al (1979). Five replicates per sample and treatment were evaluated, and the corresponding mean AE SEM was calculated.…”