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Application of capillary electrophoresis to the study of cathepsin D and protein degradation
Abstract
Capillary electrophoresis (CE) is a relatively new analytical technique, which has shown superiority in many ways over other techniques. It has been widely applied in protein, peptide, amino acid analysis. More recently, it has been widely used in biological applications, e.g. the study of protein-protein interactions, or enzyme reactions, and pharmaceutical applications. This study has employed CE for the analysis of two specific amino acids in the biological samples. 3-Methylhistidine, a post-translational product of histidine, is the indicator of in vivo protein breakdown. Capillary zone electrophoresis with acetonitrile as an additive was used to determine the level of 3-methylhistidine in less than one milligram of myofibrils. Micellar electrokinetic chromatography (MEKC) was used in the simultaneous quantitation of hydroxyproline and its isomers in collagen. Capillary electrophoretic methods were also developed for the analysis of peptidase action on proteins. Cathepsin D is an aspartyl peptidase, which plays an important role in intracellular protein digestion. It is also involved in human physiology and patho-physiology. The cathepsin D action on hemoglobin was analyzed by MEKC coupled with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein sequencing and mass spectrometry analysis. Results showed that $\sp{32}$Met-Phe$\sp{33}$ on the $\alpha$-chain was the most sensitive cleavage site of hemoglobin with a Km of 66.2 $\mu$M, and $\sp{40}$Phe-Phe$\sp{41}$ on the $\beta$-chain was the second most sensitive cleavage site. Applying MEKC to the peptide analysis, a deoxycholate buffer system showed much higher separation capacity and resolution than a sodium dodecyl sulfonate buffer system. Oregon Green labeled hemoglobin was introduced as a substrate in the peptidase assay. Using this substrate, capillary electrophoretic method with laser induced fluorescence detection achieved detection limit of 500 picograms by monitoring the production of $\alpha1$-32 peptide and fluorometric assay can measure 81 picograms of cathepsin D. This method has superiority in regards to increase sensitivity, reproducibility and availability of substrate compared to previous methods.
Subject Area
Food science
Recommended Citation
Chu, Qingyi, "Application of capillary electrophoresis to the study of cathepsin D and protein degradation" (1998). ETD collection for University of Nebraska-Lincoln. AAI9839142.
https://digitalcommons.unl.edu/dissertations/AAI9839142