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Problem. What effect will a site-specific mutation to the Pin1 protein have on catalytic efficiency? . Hypothesis. The mutations I28A and C113A will have different catalytic rates compared to regular, wild type Pin1 protein. Materials. LiCl Tetrahydrofuran (THF) Suc-AEPF-pNA HEPES

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Problem

What effect will a site-specific mutation to the Pin1 protein have on catalytic efficiency?

Hypothesis

The mutations I28A and C113A will have different catalytic rates compared to regular, wild type Pin1 protein.

Materials

  • LiCl
  • Tetrahydrofuran (THF)
  • Suc-AEPF-pNA
  • HEPES
  • Wild type Pin1
  • HCL
  • Tris-HCl
  • Cuvettes
  • Alpha-chymotrypsin
  • Stirring rod
  • Pin1 mutant form, I28A
  • Pin1 mutant form, C113A
  • UV-visible spectrophotometer
  • Gloves
  • Lab coat
  • Goggles
  • Pipettes

Procedure

  • Dissolve LiCl in THF
  • Prepare peptide solutions at 124 mM, 62 mM, 31mM, 15.5 mM, and 7.75 mM
  • Prepare assay solution consisting ofHEPES and wild type Pin1 dissolved in Tris-HCl
  • Dissolve alpha-chymotrypsin in HCL
  • Place cuvette with assay solution and chymotrypsin into spec holder of UV-visible spectrophotometer
  • Place suc-AEPF-pNA onto tip of spatula, lower into cuvette, and swirl
  • Repeat two more times for the current concentration
  • Perform at four remaining concentrations
  • Repeat with mutants I28A and C113A

A picture of Connor Burke placing the assay solution into the cuvette for the chromogenic assay

Figure 1. A flow diagram that outlines the basic procedure of the chromogenicassay