A Simple and Sensitive Inhibitory Kinetic Method for the Carbocisteine Determination

A fast, reproducible, and sensitive method is proposed for the kinetic determination of carbocisteine (CCys). The method depends on the inhibitory property of carbocisteine, which reduces the Hg2+ catalyzed substitution rate of cyanide from [Ru(CN)6]4- with N-R-salt (1-Nitroso-2-naphthol-3,6-disulfonic acid disodium salt) via forming a stable complex with Hg2+. Spectrophotometric measurements were carried out by recording the absorbance at 525 nm (λmax of [Ru(CN)5 Nitroso-R-Salt]3- complex) at a fixed time of 10 and 15 min under the optimized reaction conditions with [N-R-salt] = 4.5 × 10-4 M, I = 0.05 M (KNO3), Temp = 45.0 ± 0.2 o C, pH = 7.0 ± 0.03, [Hg2+] = 8.0 × 10-5 M and [Ru(CN)64-] = 4.25 × 10-5 M. With the proposed method, CCys can be determined quantitatively down to 3.0 × 10-6 M. This methodology can be effectively used for the rapid quantitative estimation of CCys in the pharmaceutical samples with good accuracy and reproducibility. The addition of common excipients in pharmaceuticals even up to 1000 times with [CCys] does not interfere significantly in the estimation of CCys. Resumen. Se propone un método rápido, reproducibley sensible para la determinación cinética de la carbocisteina (CCys). El método depende de la propiedad inhibitoria de la carbocisteina que reduce la tasa de sustitución catalizada por Hg2+ del cianuro de [Ru(CN)6]4- con la sal N-R (sal disódica del ácido 1-Nitroso-2-naftol-3,6-disulfónico) mediante la formación de un complejo estable con Hg2+. Las mediciones espectrofotométricas se llevaron a cabo registrando la absorbancia a 525 nm (λmax del complejo [Ru(CN)5 Sal-Nitroso-R]3-) en un tiempo fijo de 10 y 15 min en las condiciones de reacción optimizadas con [sal-NR] = 4.5 × 10-4 M, I = 0.05 M (KNO3), Temp = 45.0 ± 0.2 o C, pH = 7.0 ± 0.03, [Hg2+] = 8.0 × 10-5 M y [Ru(CN)64-] = 4.25 × 10-5 M. Con el método propuesto, CCys se puede determinar cuantitativamente hasta 3,0 × 10-6 M. Esta metodología se puede utilizar eficazmente para la estimación cuantitativa rápida de CCys en las muestras farmacéuticas con buena precisión y reproducibilidad. La adición de excipientes comunes en productos farmacéuticos incluso hasta 1000 veces con [CCys] no interfiere significativamente en la estimación de CCys.

Isopeptidase Kinetics Determination by a Real Time and Sensitive qFRET Approach

Isopeptidase activity of proteases plays critical roles in physiological and pathological processes in living organisms, such as protein stability in cancers and protein activity in infectious diseases. However, the kinetics of protease isopeptidase activity has not been explored before due to a lack of methodology. Here, we report the development of novel qFRET-based protease assay for characterizing the isopeptidase kinetics of SENP1. The reversible process of SUMOylation in vivo requires an enzymatic cascade that includes E1, E2, and E3 enzymes and Sentrin/SUMO-specific proteases (SENPs), which can act either as endopeptidases that process the pre-SUMO before its conjugation, or as isopeptidases to deconjugate SUMO from its target substrate. We first produced the isopeptidase substrate of CyPet-SUMO1/YPet-RanGAP1c by SUMOylation reaction in the presence of SUMO E1 and E2 enzymes. Then a qFRET analyses of real-time FRET signal reduction of the conjugated substrate of CyPet-SUMO1/YPet-RanGAP1c to free CyPet-SUMO1 and YPet-RanGAP1c by the SENP1 were able to obtain the kinetic parameters, Kcat, KM, and catalytic efficiency (Kcat/KM) of SENP1. This represents a pioneer effort in isopeptidase kinetics determination. Importantly, the general methodology of qFRET-based protease isopeptidase kinetic determination can also be applied to other proteases.

Quantitative Estimation of D-Penicillamine in Pure and Pharmaceutical Samples Using Inhibitory Kinetic Spectrophotometric Method

Sulfur is the key element in a large number of drugs and bioactive molecules. Organo-sulfur compounds inhibit the catalytic efficiency of Hg2+ by forming a stable complex with it. The Hg2+ catalyzed exchange rate of cyanide with pyrazine from [Ru(CN)6]4- will be reduced by the addition of the sulfur-containing drug, D-penicillamine (D-PCN). This inhibitory property of D-PCN can be employed for its micro-level kinetic determination. Optimum reaction condition viz. Temperature = 45.0 ± 0.1 o C, I = 0.1 M (KCl), [Hg+2] = 1.5 × 10-4 M, [pyrazine] = 7.5 × 10-4 M, pH = 4.0 ± 0.02, and [Ru(CN)64-] = 5.25 × 10-5 M were utilized to investigate the kinetic measurements at 370 nm (λmax of [Ru(CN)5 Pz]3- complex). To acknowledge the inhibition induced by D-PCN on Hg2+ catalyzed substitution of cyanide with pyrazine from [Ru(CN)6]4-, a modified mechanistic scheme has been proposed. D-PCN can be quantitatively determined up to 1.0 × 10-6 M level by the proposed analytical method. The methodology can be economically and effectively employed for the quantitative determination of D-PCN in different samples. This methodology can also be convincingly adopted for the quick determination of D-PCN in the pharmaceutical samples with good accuracy and reproducibility. The addition of common excipients in pharmaceuticals even up to 1000 times with [D-PCN] does not interfere significantly in the estimation of D-PCN.

Micro-level Estimation of Methionine Using Inhibitory Kinetic Spectrophotometric Method

A large number of bioactive molecules and drugs contain sulfur as an important constituent. Organo-sulfur compounds form a stable complex with Hg2+, thereby inhibiting its catalytic activity. The Hg2+ catalyzed the exchange rate of cyanide with nitroso-R-salt [N-R-salt] from [Ru(CN)6]4- will be reduced by the addition of sulfur-containing amino acid, methionine (MET). This inhibitory property of MET can be employed for its micro-level kinetic determination. Optimum reaction condition viz. I=0.05 M (KNO3), pH = 7.0 ± 0.02, [Ru(CN)64] = 5.25 × 10-5 M, [N-R-salt] = 6.5 × 10-4 M, [Hg+2] = 5.5 × 10-5 M, and Temperature = 45.0 ± 0.1 o C were utilized to investigate the kinetic measurements at 525 nm (λmax of [Ru(CN)5 N-R-salt]3- complex). To explain the mechanism of inhibition caused by methionine on Hg2+ catalyzed exchange of cyanide with N-R-salt from [Ru(CN)6]4-, a modified mechanistic scheme has been proposed. MET can be quantitatively determined up to 2.5 × 10-6 M level by the proposed analytical method. The methodology can be economically and effectively employed for the quantitative estimation of MET in distinct samples.