Extraction, purification, and biochemical characterization of serine protease from leaves of Abrus precatorius.

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ∼ 28 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co2+, Ni2+, Hg2+, and Zn2+ while its activity has increased in the presence of Ca2+ and Mg2+. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its Vmax and Km determined using casein as a substrate were 94.34 U/mL and 349.07 µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.

Rapid detection of abrin in foods with an up-converting phosphor technology-based lateral flow assay.

Abrin is a natural plant toxin found in the seeds of Abrus precatorius. It may be used for food poisoning or bioterrorism, seriously endangering public health. In this study, a reliable method for the rapid detection of abrin in foods was developed, based on an up-converting phosphor technology-based lateral flow assay (abrin-UPT-LFA). Nine high-affinity monoclonal antibodies (mAbs) against abrin were prepared, and the optimum mAbs (mAb-6F4 and mAb-10E11) were selected for use in the assay in double-antibody-sandwich mode. The assay was confirmed to be specific for abrin, with a detection sensitivity of 0.1 ng mL-1 for standard abrin solutions. Good linearity was observed for abrin quantitation from 0.1 to 1000 ng mL-1 (r = 0.9983). During the analysis of various abrin-spiked food samples, the assay showed strong sample tolerance and a satisfactory limit of detection for abrin (0.5-10 ng g-1 for solid and powdered samples; 0.30-0.43 ng mL-1 for liquid samples). The analysis of suspected food samples, from sample treatment to result feed-back, could be completed by non-professionals within 20 min. Therefore, the abrin-UPT-LFA is a rapid, sensitive, and reliable method for the on-site detection of abrin in foods.

Purification and dose-dependent toxicity study of abrin in swiss albino male mice.

Abrin, a phytotoxin obtained from the seeds of the Abrus precatorius plant, is highly toxic with an estimated human fatal dose of 0.1­1 µg/kg. In this study, abrin was purified and characterized through SDS PAGE and mass spectrometry analysis; further study on toxicity was carried out to investigate the alteration in biochemical, and hematological variables through histopathological observations in mice. The intraperitoneal LD50 value of purified abrin for mice was found to be 0.91µg/kg of body weight. Mice were exposed to 0.4 and 1.0 LD50 abrin doses intraperitoneally and observed on days 1, 3, and 7. Plasma GOT and GPT levels increased significantly at both doses. At 1.0 LD50 dose, alkaline phosphatase, bilirubin, urea, uric acid, and creatinine levels increased, whereas albumin, total protein, glucose and cholesterol levels decreased significantly. Abrin intoxication also altered the hemoglobin, WBC, and RBC counts significantly at 1.0 LD50 dose. Liver GSH levels decreased while lipid peroxidation increased significantly in a dose­dependent manner. Biochemical changes were supported by the histological investigation, which also showed the degenerative changes in organs. In conclusion, abrin intoxication caused toxic effects and severe damages on studied organs mediated through alteration in biochemical and hematological variables, lipid peroxidation, and degeneration.

Effect of fluidic transport on the reaction kinetics in lectin microarrays.

Lectins are the proteins which can distinguish glycosylation patterns. They are frequently used as biomarkers for progressions of several diseases including cancer. As the lectin microarray based prognosis devices miniaturize the process of glycoprofiling, it is anticipated that their performance can be augmented by integration with microfluidic framework. This is analogous to microfluidics based DNA arrays. However, unlike small oligonucleotide microarrays, it remains uncertain whether the binding reaction-kinetic parameters can be considered invariant of imposed hydrodynamics, for relatively larger and structure sensitive molecules such as lectins. Here we show, using two standard lectins namely Concanavalin A and Abrus Agglutinin, that the steady state binding efficiency unexpectedly declines beyond a critical shear rate magnitude. This observation can be explained only if the associated reaction constants are presumed to be functions of hydrodynamic parameters. We methodically deduce the shear rate dependence of association and dissociation constants from the comparison of experimental and model-simulation trends. The aforementioned phenomena are perceived to be the consequences of strong hydrodynamic perturbations, culminating into molecular structural distortion. The exploration, therefore, reveals a unique coupling between reaction kinetics and hydrodynamics for biomacromolecules and provides a generic scheme towards futuristic microfluidics-coupled biomedical assays.