Questions 61 - 71 ELMA design

61. Buffer Making What would be the most practical way of making the Tris solution Weigh out 2.4228 g of Tris, dissolve in 20 ml of water, then adjust the pH to 8.1 by adding concentrated HCl Dissolve 2.4228 g of Tris in 20 ml water, then adjust the pH to 8.1 by adding dilute HCl Dissolve 2.4228 g of Tris in 10 ml water, then adjust the pH to 8.1 with HCl, then add water to 20 ml Dissolve about 2.5 g of Tris in about 10 ml water, adjust the pH to 8.1 with HCl, then add water to give a volume equal to 20 x (weight taken/2.4228) Accurately measure 20 ml water and adjust its pH to 8.1 with HCl, then add 2.4228 g of Tris

62. Using a Stock Solution To achieve the above, how much of the stock acetyl-CoA solution (as recommended by your assistant) should you add to the cuvette? 120 µl 8.2 µl 1.2 µl 100 µl 2.5 µl

63. Absorbance to nmol If the absorbance change was 0.75 per min, how fast is the AMOUNT of yellow product increasing in the cuvette? 75 µmol per min 50 nmol per min 5 nmol per min 15 nmol per min 15 µmol per min

64. Small Volume Additions You decide that you need 50 mU of citrate synthase (CS) in the cuvette. What is the MOST PRACTICAL way of doing this? Adding 0.025 µl of the stock CS directly to the cuvette Adding 25 µl of the stock CS directly to the cuvette Mixing 1 µl of the stock CS with 999 ul Tris buffer and then adding 25 ul of this diluted CS to the cuvette Mixing 10 µl of stock CS with 990 ul Tris buffer and then adding 2.5 ul of this diluted CS to the cuvette Adding 2.5 µl of the stock CS directly to the cuvette

65. Rapid Colour Change What is NOT a plausible interpretation of these observations? Something in the citrate synthase stock has a lot of free thiol groups The acetyl CoA solution that you made up has partially degraded to give CoA You have added a lot more citrate synthase than you thought The DTNB spontaneously degrades at the pH of the Tris Your acetyl CoA solution contains some free thiol groups

The Time Course After some trouble-shooting, you have convinced yourself that you can now make the citrate synthase assay work. The scheme that you have come up with is to set up a cuvette containing 20 mM TRIS, 1 mM EDTA, 0.1 mM DTNB, 0.1 mM acetyl-CoA and 50 mU of citrate synthase (total volume of 1 ml). Then you mix the cuvette contents, put it in the spectrophotometer and zero its absorbance. Then you add 0.05 mM oxaloaceate and record the absorbance change with time. Over the first 30 seconds, you find that the absorbance rises by about 0.35.

66. Limiting Factors To make this system measure acetyl-CoA, you replace the stock acetyl-CoA with a volume of an ‘unkonwn’ sample (eg, a tissue extract) Which statement is the MOST LIKELY prediction of what will happen next? The rate of increase in absorbance will remain constant for at least 10 minutes The absorbance will plateau when the acetyl-CoA runs out The rate will fall after about 5 minutes as the citrate synthase becomes degraded The absorbance will fall as the all the DTNB gets used up In less than five minutes all the oxaloacetate will run out

67. ELMA principles What else do you have to do to make this system useful as an assay for acetyl‑CoA? Ensure that oxaloacetate is never in excess Ensure that DTNB is in excess Ensure that citrate synthase does not drive the reaction to completion in less than 5 minutes Ensure that the absorbance of the system plateaus above 2 Ensure that the unknown acetyl-CoA is added in excess

68. Role of Unknown Chemical How does the Triton X-100 and freezing/thawing help you get the most accurate results from the tissue samples? It activates citrate synthase It increases the release of citrate synthase from the mitochondrial matrix It causes the hydrolysis of acetyl-CoA It increases the release of metabolites from the cell It increases the turbidity of the extract

69. Tissue Extracts When you use the real tissue extract, you anticipate problems will occur. Which is NOT likely to be a cause of such problems?. Tissue extracts can contain pigments that absorb in the visible range Tissue extracts may contain CoA Tissue extracts contain citrate synthase The acetyl CoA concentration in the tissue extracts may be too low Tissue extracts can be cloudy

70. Back Calculation If the acetyl-CoA concentration of a sample of heart homogenate is found to be 50 µM, what is the concentration of acetyl-CoA in the tissue? 1000 nmol g-1 50 nmol g-1 10 nmol g-1 5000 nmol g-1 100 nmol g-1

71. Spotting Good Data What would give you the most confidence that your acetyl-CoA assay was working well? Each tissue extract gives a different final result for [acetyl-CoA] Different volumes of the same tissue extract plateau at the same absorbance The final result for [acetyl-CoA] for each tissue extract is identical For a particular tissue extract, the plateau absorbance is proportional to the amount of extract added The absorbance of each assay plateaus at the same value