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Nucleophile Concentration Experiment

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Chemical reactivity measurement of test chemicals towards nucleophiles is getting more attention as an alternative to animal method in testing for potency of skin sensitizers. In view of this, alternative methods are expected to be highly reproducible. To achieve this, there is need for proper investigation of appropriate method of analysis. Depletion of protein nucleophiles and formation of covalent adducts between skin sensitizers and dermal proteins are very important processes in skin sensitization. These are monitored and detected through various means like ultraviolet-visible spectrophotometry, high performance liquid chromatography/mass spectrometry (HPLC-MS), liquid chromatography/mass spectrometry (LC-MS), nuclear magnetic resonance/mass spectrometry (NMR-MS), etc. There is increase in the use of mass spectrometry to determine the extent of protein modification and the exact location. The method to be adopted in the detection of hapten-protein adducts depends on the sensitivity of the technique. …show more content…

Compound 1 contained no reactive site, so no reaction was expected to take place. The other two contained epoxides and therefore they were expected to react. A solution of each compound was added to hexapeptide and the resulting solution was analyzed using HPLC-MS (positive ESI mode). Adducts formation were observed in compounds with epoxide group while spectrum did not indicate any adducts in compound with only diene structure [32]. HPLC-MS was used to confirm MBT-cysteine adduct formed through a disulfide bridge after reduction of thiol of cysteine molecule by MBTS. The model protein used was Bovine Serum Albumin (BSA) to evaluate direct haptenation between MBTS and Cys34 on BSA which lead to disulfide formation. Potential importance of disulfide formation was highlighted as a general haptenation and protein modification pathway

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TLC was used to identify the actual unknown product as well as other products/reactants present in the filtered solution. The procedure was conducted by placing a TLC plate in a developing chamber that is filled with a small amount of solvent. The solvent cannot be too polar because it will cause spotted compounds on the TLC plate to rise up too fast, while a very non-polar solvent will not allow the spots to move. The polarity of the spots also determines how far it moves on the plate; non-polar spots are higher than polar ones. After spots on the TLC form, the Rf values are calculated and used to analyze the similarity of the compounds.

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1.1 Abstract The purpose of quantitative analysis of protein using a spectrophotometer is to measure the concentration of proteins in a given sample. The experiment is conducted by laboratory method (Biuret Test) and using spectrophotometer to analyze the absorbance of reactants at 540 nm, hence determining the concentration of the proteins in a given sample. The purpose of stopped enzyme assay to study B-galactosidase is to determine the effect of temperature and concentrations of substrate on enzyme activity.

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