BIOLOGY 371

INDEPENDENT RESEARCH

WEEK 10

Divalent Metal Requirement

1. Does IDH require a divalent metal ion for activity?
2. What are the effects of assorted heavy metals on IDH activity?
3. Provide the full chemical reaction of divalent metal ion function.
4. Model heavy metal toxicity on enzyme function.
5. Design the next [metal ion] experiment that we should preform.

Procedure

1. Prepare 500 mL of IDH assay buffer with no divalent metal ion.
2. Prepare 1 mL of concentrated divalent metal ion solutions. Concentrations should be such that 10 m l in 1 mL of assay solution will yield a final concentration of 2 mM. Use MgCl2 as the standard divalent ion.
3. Prepare an enzyme solution so that 10 m L will give a slope of about 0.8, using standard assay conditions.
4. To 1 mL of assay solution with no metal ion, add 10 m l of concentrated MgCl2 solution. Do triplicate assays.
5. Next, determine activity without adding the divalent metal ion.
6. Then, determine activity replacing Mg2+ by adding 10 m l of concentrated solutions of: Mn2+, Ca2+, Cd2+, Cu2+, Hg2+, Ni2+, Sr2+ and Zn2+.
7. Initiate assays using standard conditions and allow to proceed for 1.5 min.

Now add 10 m l of concentrated solutions of the 9 divalent metal ions.
Allow the reaction to proceed for 2 more min.

8. Summarize your results.
9. Design the next experiment concerning metal ion interactions with IDH.

[What is the effect of making the assay solution 2 mM EDTA?]

METAL ION SOLUTIONS


1. Prepare concentrated* solutions [1 mL] of divalent metal ions such that the addition of 10 m l to 1 mL of assay solution will result in a 2 mM final concentration. Perform triplicate assays for each and compare the effects of the various metals on IDH activity.

 Metal  Mol Weight  Gm/mL*
 Manganese    
 Magnesium    
 Calcium    
 Cadmium    
 Copper    
 Mercury    
 Nickel    
 Strontium    
 Zinc    

* Remember - these concentrations need to be 100-fold more concentrated than the concentration in the assay solution.

Figure 16. Km Mg2+ and Mn2+ for isocitrate dehydrogenase fromBrassica hirtsa.

Return to Schedule

© Copyright 2000 Department of Biology, Davidson College, Davidson, NC 28036
Send comments, questions, and suggestions to: jowilliamson@davidson.edu