Biochemistry Lab Assay PDF

Preview

Summary

Assay Description Assigment...

Description

Biochemistry Lab Assay Description Assignment Natalie Yam September 25, 2016

1. A colormetric assay for UNH (Uridine Nucleocide-Ribohydrolase) is used to measure the activity of this enzyme. UNH is allowed to react with its substrate, uridine, for a certain time period. UNH catalyzes the breakage of the N-glycosidic bond in uracil; the bond located between the carbon of ribose and the nitrogen on uracil. The product of this reaction is a free nucleobase and a ribose. To stop the reaction, a solution of copper sulfate is added. It provides Cu 2+ ions for the ribose to reduce into Cu+ ions, which then forms a complex with a molecule called neocuproine. The solution is then heated in a water bath for the yellow color to develop. This assay measures product formation so the presence of a yellow color means that ribose has formed. The more yellow the color, the more ribose has been formed. The absorbance at 450 nm is recorded to determine its activity. Uracil, after glycosidic bond cleaved

Ribose, after glycosidic bond cleaved

2. To measure the formation of ribose, a reducing sugar, through the catalytic cleaving effects of UNH, a colormetric assay was performed. This was performed through 2 mM of uridine in 50mM MES at pH 6.2, containing 0.3M KCl. The assay volumes are 1 mL. The addition of 0.3 mL of a reagent stock solution containing 4% Na 2CO3, 1.6% glycine, 0.045% CuSO4 · H2O, and 0.3 mL of 0.12% 2,9-dimethyl-1,10- phenathroline, with a total volume of 1.6 mL, terminated the reaction. The solution is heated in a hot water bath at 95 ˚F for eight minutes. The UV-VIS wavelength used for this detection is 450 nm.

The fraction that contained protein was put through electrophoresis in polyacrylamide gels containing SDS to estimate purity. 20 μ Uridine g UNH, and Protein Plus Kaleidoscope

standards (BioRad) were loaded onto the gel and electrophoresis was ran. The enzyme was then flash frozen in a -80  C ethanol bath and stored at -80 C....