CH 150: Introduction to Biochemistry

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Spectrophotometry:

Additivity of Spectra

Objective: The objective of this experiment is to determine the additivity of spectra.

 Experimental Procedure:

 Reagents:

       0.10 M Tris-Cl buffer, pH 8.0

            0.050 mM DNP-glutamate(N-2,4-dinitrophenyl-L-glutamine) in buffer

            0.010 mM cytochrome c

1.      Prepare 4 mL of each of the following solutions using 0.1 M Tris-Cl buffer to dilute where appropriate:

                     (1)   Solution 1: 25 mM DNP-glutamate

2 mL 0.050 mM DNP-glutamate

2 mL 0.10 M Tris-Cl buffer, pH 8.0

        (2)   Solution 2: 5 mM cytochrome c

2 mL 0.010 mM cytochrome c

2 mL 0.10 M Tris-Cl buffer, pH 8.0

                    (3) Solution 3: 25  mM DNP-glutamate + 5 mM cytochrome c

                        2 mL 0.050 mM DNP-glutamate

                        2 mL 0.010 mM cytochrome c

2.      Using the Tris-Cl buffer as a blank, determine the absorbance spectra of solutions 1, 2, and 3 from 350 to 500 nm at 10 nm intervals.  Remember that you must zero the absorbance with the blank at each new wavelength.

·        Plot A: Plot absorbance (y-axis) versus wavelength (x-axis) for each solution (all three spectra should appear on the same plot).

3.      Subtract the spectra of solution 1 from solution 3.

·        Plot B: Plot the resulting spectrum (solution 3 - solution 1) and the spectrum of solution 2.  The two spectra should appear on one graph to facilitate comparison.

4.      Also subtract the spectrum of solution 2 from solution 3.

·        Plot C: Plot the resulting spectrum (solution 3- solution 2) and the spectrum of solution 1 together on another graph.

What can you conclude from these results?

5.      You will be given an unknown solution containing DNP-glutamate and cytochrome c.  Measure the absorbance of the solution at the appropriate wavelength(s) (the lmax for DNP-glutamate and cytochrome c).

Data Tables

Unknown: Letter _____________

l1 = _______________                 Abs @ l1 = _______________

l2 = _______________                 Abs @ l2 = _______________

Questions:

 1.      At what wavelength does DNP-glutamate have its maximum absorbance? (In other words, what is the lmax for DNP-glutamate?)

___________________ nm

2.      What is the l-max for cytochrome c?

___________________ nm

3.      Calculate the extinction coefficients(E) at l_max in units of mM^-1 cm^-1, mM^-1 cm^-1, and M^-1cm^-1 for DNP-glutamate and cytochrome c from the spectra of solutions 1 and 2, respectively.

DNP-glutamate                                                         cytochrome c

E =   ____________ mM^-1 cm^-1                   E =   ____________ mM^-1 cm^-1

   =   ____________ mM^-1 cm^-1                      =   ____________ mM^-1 cm^-1

   =  _____________ M^-1 cm^-1                        =  _____________ M^-1 cm^-1

4.      Look at Plots B and C.  What can you conclude from your results?

5.      What is the extinction coefficient of DNP-glutamate at l2 (lmax for cytochrome c)? What is the extinction coefficient of cytochrome c at l1 (lmax for DNP-glutamate)?

DNP-glutamate at l 2                                   cytochrome c at l 1

E =   ____________ mM^-1 cm^-1                  E  =   ____________ mM

6.      What is the concentration of DNP-glutamate and cytochrome c in your unknown?  Show your calculations below.

[DNP-glutamate] = ________ mM                    [cytochrome c] = ________ mM