Preparation of standard curve

Materials

• 8 mM DOPA
• Enzyme Extract - From Exercise 5.1
• Test tubes
• 5 ml Pipette
• 0.1M Citrate Buffer, pH 4.8
• Spectrophotometer and Cuvettes

Procedure

1. Begin by preparing a standard solution of the orange colored dopachrome from L-DOPA. To 10 ml of 8 mM DOPA, add 0.5 ml of your enzyme extract 4 and allow the solution to sit for 15 minutes at room temperature. During this period, all of the DOPA will be converted to dopachrome, and your solution will now contain 8 mM dopachrome. Dopachrome is somewhat unstable in the presence of light and should be stored in an amber bottle or out of the light.

2. Prepare a 1:1 series of dilutions of the 8 mM Dopachrome to yield the concentrations in the following table: Add 3.0 ml of each indicated concentration to tubes #1-8.

   Tube #	Final Concentration of Dopachrome (mM)
   1	0
   2	0.125
   3	0.25
   4	0.5
   5	1.0
   6	2.0
   7	4.0
   8	8.0

   With these dilutions, you have prepared tubes containing concentrations from 0 to 8 mM dopachrome (tubes 1-8). Tube 1 contains no dopachrome and is used for blanking the spectrophotometer.

   The units of concentration are millimolar (mM). A 1.0 mM solution contains .001 moles per liter or .000001 moles per ml. Thus, with a volume of 3.0 ml, there are .000003 moles of dopachrome, or 3 micromoles. Correspondingly, tubes 2-8 contain 1 to 24 micromoles of dopachrome. For the remainder of this exercise, be sure to distinguish between concentration (mM) and total amount of substance present (micromoles).

3. Turn on your spectrophotometer and set a wavelength of 475. Use tube #1 from the above dilutions as a blank and adjust the spectrophotometer for 0 and 100% T. Read the absorbance (or read and convert transmittance) of each of the solutions in tubes 2-8 and complete the following table:

   Tube	Concentration of Dopachrome (mM)	Absorbance	A/C
   #1	0	0	----
   #2	0.125
   #3	0.25
   #4	0.5
   #5	1.0
   #6	2.0
   #7	4.5
   #8	8.0

4. Calculate the values for the last column of the table. This column represents the simplest calculation of the extinction coefficient for dopachrome absorbance. Average the values in this column and enter the number at the bottom of the column. This is the average extinction coefficient and can be used in subsequent determinations of dopachrome concentrations according to the Beer-Lambert law.

   You can more accurately determine the extinction coefficient by performing a linear regression analysis of your data, and computing the slope and y intercept. The slope of the linear regression will represent the extinction coefficient for your sample.

5. Plot a scattergram of the absorbance value against the concentration of dopachrome. The known concentration of dopachrome should be the x axis, while absorbance should be the y axis.
   

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