Progress 03/22/04 to 12/31/04
Outputs
4d Progress report. This report serves to document research conducted under a Non-funded Cooperative Agreement between ARS and the University of Illinois. Additional details of research can be found in the report of the parent Cooperative CRIS 3620-41000-119-00D, "Nonfood Utilization of Cereal and Soy Based Co-Products". The research was terminated December 2004. A new system was devised to solubilize zein, which is a water insoluble protein, with a surfactant, Rapi Gest, that does not denature either the zein or the enzymes used for protelysis. Proteolysis was performed with three enzymes: trypsin, chymotrypsin, Alcalase on two different commercial sources of zein including Freeman zein and Showa Japanese white zein. Our findings with sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated that zein was completely proteolyzed to peptides. Molecular weights of those peptides were determined by MALDI- TOF MS (mass spectral) analysis performed by
research at University of Illinois, Champaign-Urbana. Based on the known amino acid sequence for a- zein, primary component of corn zein, in combination with MALDI-TOF analyses; the enzymes trypsin and chymotrypsin, each with known cleavage sites, proteolyzed the a-zein to the correct number of peptides. However, discrepancies in molecular weights of theoretical versus actual led us to believe that some of the peptides interacted with each other to yield a complex system that would require separation, isolation of purified peptides and sequencing to identify the resulting polypeptides. Our original objective was to enzymically proteolyze zein to polypeptides that would be investigated as ACE (angiotensin converting enzyme) inhibitors that are used to lower blood pressure. This research was terminated because Japanese researchers had published similar work with zein proteolyzed with thermolysin to generate polypeptides some of which were found to possess ACE inhibitor activity and
further investigation would be deemed inconsequential.
Impacts
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Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? D. Progress report. This report serves to document research conducted under a Non-funded Cooperative Agreement (NFCA) between ARS and Illinois Corn Marketing Board (ICMB). Additional details of research can be found in the report of the parent research project #3620-41000-105-00D, Chemistry and Processing of Cereal and Soy based Co-products for Non-food Utilization. A new system was devised to enzymically proteolyze zein into low molecular weight peptides with a surfactant, RapiGest, that did not denature the proteins. Proteolysis was performed with three enzymes, trypsin, chymotrypsin, and Alcalase. Based on our findings with sodium dodecyl sulfate - polyacrylamide gel electrophoresis, zein was completely proteolyzed to peptides whose respective molecular weights were then determined by MALDI-TOF MS analysis. Based on the known amino acid sequence of alpha zein, the primary component of corn zein,
in combination with MALDI-TOF analyses, the enzymes trypsin and chymotrypsin, each with known cleavage sites, proteolyzed the alpha zein to the correct number of peptides. However, discrepancies were found between theoretical molecular weights of peptides versus our actual findings. Because of this discrepancy the identification of molecular weights and identification of amino acid sequence of the individual peptides requires isolation of each of the peptides. Our objective to isolate individual peptides for their physiological evaluation has yet to be determined.
Impacts
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