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The development of a solid phase screening assay for the detection of dipeptide synthesis Yue, Eliza

Abstract

A rapid screening assay was developed to detect formation of DNP-asn-leu dipeptides on a special type of microtiter plate, called the Nunc Immuno Module. Secondary amino groups, to which small molecules can be coupled, were grafted onto the surface of the plate. The carboxyl group on L-leucine molecules, activated in the presence of 0.1 M N-hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, were covalently coupled to these amino groups; 11.04 fxq of L-Leucine was used in the coupling reaction. The reaction mixture, consisting of either commercial grade thermolysin or crude protease mixture isolated from Bacillus subtilis culture, DNP(dinitrophenyl)-L-asparagine and pH 6 sodium acetate buffer solution, was subseguently added to the wells. During incubation at 48°C, dipeptide formation occurred between the immobilized leucine and the free DNP-L-asparagine in the liquid phase. After washing off unreacted DNP-L-asparagine, the final product was detected by the addition of an antibody-peroxidase conjugate, which reacted specifically with the dinitrophenyl group attached to the amino group of the asparagine molecule. The amount of increase in optical density measured at 492 nm was proportional to the relative amount of dipeptides formed on the microtiter plate. Two-way analysis of variance showed that the effect of enzymatic treatment on the mean optical density measured at 492 nm was significant (P

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