In lab for this semester, we isolated LDH from beef heart homogenate. However, I

Question

In lab for this semester, we isolated LDH from beef heart homogenate. However, I still don't have any idea on what we should have seen with regards to activity in each protein assay. This is a summary of what we did for each lab and the tables that follow. I really just need help understanding which of the data in my tables are right and which should have different numbers (some assays shouldn't have activity).

Enzyme Purification: Ammonium Sulfate. Beef heart homogenate was obtained and centrifuged at 20000 x g for 15 minutes (consistent with all other centrifugations). Ground ammonium sulfate was added at .230 g per milliliter of supernatant creating a 40% ammonium sulfate cut. The 40% cut pellet was resolubilized with 2 mL of phosphate buffer and the 40% supernatant centrifuged. 166 g of ground ammonium sulfate was added per milliliter of supernatant, creating the 65% cut. The solution was centrifuged. The supernatant was collected, and the pellet was resuspended in 5 mL of phosphate buffer and then placed into a Slide-a-Lyzer and dialyzed in .03 M bicine.

          Analysis of Fractions. A reaction cocktail was created which contained 1.9 mL CAPS buffer (0.14M, pH 10), 0.5 mL NAD+ (6mM), and 0.5 mL of lactate (0.15M, pH 10) and added to each fraction so that each assay had a total volume of 2.9 mL. 10µL of enzyme mixed with 90 µL of water was added to the assay. The Spec 20 was then set to 340 nm and used to obtain the change in absorbency of each assay per minute.

          Enzyme Purification: Column Chromatography. The Q-sepharose was ran with 0mM NaCl buffer (flow through fraction). A step gradient was then ran with 20 mL of NaCl buffer that increased by .2M every time the buffer was added, starting with .2M. The Spec 20 was set up at 280 nm to obtain absorbencies of each fraction. The pooled fraction is created by adding samples with the most activity.

          Protein Concentration Analysis. 5 dilute samples of 1.5 mL of varying BSA concentrations were made. 1.5 mL Coomassie reagent was then added to each sample. The Spec 20 was set at 595 nm to obtain absorbencies of each sample. This procedure was then repeated with the protein samples.

          Enzyme Kinetics. The reaction cocktail was made and included a volume of LDH in which the change in absorbency per minute was between .15 and .25. Varying amounts of NAD+ was then added to 2.15 mL CAPS buffer, .5 mL lactate, and .10 mL (water + enzyme). The Spec 20 was then set to 340 nm and absorbencies were recorded for each mixture with varying NAD+ amounts.

  

These are the absorbancies calculated for the first lab. I'm not sure which fractions should have activity and which ones shouldn't.

Here is a breakdown of everything. Again, I'm not sure what's right and what's wrong. if you can help me out, it would be greatly appreciated.

Time (min) 20000 x g supernatant 40% supernatant 40% pellet 65% supernatant 65% pellet 65% dialysis pooled 0.000 0.200 0.090 0.079 0.161 0.201 0.123 0.047 0.167 0.267 0.156 0.086 0.138 0.430 0.225 0.073 0.333 0.321 0.201 0.082 0.122 0.544 0.327 0.094 0.500 0.362 0.243 0.079 0.104 0.665 0.387 0.114 0.667 0.399 0.301 0.076 0.091 0.772 0.452 0.135 0.833 0.437 0.320 0.073 0.085 0.845 0.514 0.158 1.000 0.470 0.357 0.071 0.085 0.934 0.573 0.175 1.167 0.501 0.392 0.077 0.088 1.040 0.621 0.194 1.333 0.534 0.422 0.082 0.090 1.075 0.671 0.217 1.500 0.564 0.451 0.084 0.092 1.144 0.720 0.233 1.667 0.591 0.485 0.084 0.094 1.179 0.770 0.251 1.833 0.618 0.510 0.085 0.097 1.234 0.798 0.265 2.000 0.642 0.541 0.085 0.098 1.281 0.839 0.281 2.167 0.669 0.567 1.351 0.881 0.298 2.333 0.690 0.590 1.398 0.913 0.314 2.500 0.709 0.616 1.403 0.945 0.328

Explanation / Answer

The data you presented here are correct.

The 20000g supernatant will have the activity due to the presence of proteins

40% pellet will have more protein than the 40% supernatant as most of the protein will be pelleted out.

65% supernatant i.e third round of pritein precipitation, will totally precipitate the protein leaving no protein in the supernatant due to which you dont get any activity. Most of the activity is present in the pellet

When you dialyse the 65% pellet, the protein activity increases due to concentration of the protein.

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