The overarching goal of this experiment was to isolate and purify lactate dehydrogenase, an enzyme of significant importance in mammalian metabolism, and eventually determine the identity of the particular isozyme, which was either heart or muscle tissue. Essentially, with each subsequent purification step, the corresponding purification intermediate was expected to result in the loss of LDH enzyme at a lower rate compared to total protein. Ultimately, the significant isozyme contributor to the unknown sample was identified to be LDH-4, an isozyme with subunit composition HMMM.
The identification of the particular isozyme was made using the native gel electrophoresis results, supplemented by SDS-PAGE. The results from the native gel electrophoresis are depicted in Figure 7. Using the results from the native gel alone, it is observed that both the affinity and size exclusion purified LDH resulted in streaking bands, which had corresponding bands at each LDH standard and bands located in between the two. Since the muscle tissue LDH has an isoelectric point of 8.18, while heart tissue LDH has an isoelectric point of 5.57 relative to a pH environment of 9.5, the heart tissue travels further down the gel due to its larger negative charge. From this, two different conclusions can be inferred: (1) On one hand, the identity of the isozyme can be attributed to the darkest band and its corresponding LDH standard, while the rest of the downstream streaking bands can be attributed to contaminating proteins. In this experiment, the resulting darkest bands were located higher up in the gel, while the rest of the bands downstream got sequentially lighter. Using this logic, one would conclude the muscle tissue as the significant contributing isozyme. (2) On the other hand, the identity of the isozyme can be attributed to a hybrid mixture, disregarding the preceding conclusion’s assumption that the intermediary bands were contaminating proteins. This conclusion dictates that the major isozyme contributor is muscle type LDH, due to the presence of darker bands higher up in the gel which corresponds to the muscle type LDH standard, with partial contribution from heart type LDH, which is represented by the progressively lighter bands in between both standards and at the standard corresponding to heart type LDH. The use of SDS-PAGE aided in determining which conclusion to follow.
The results of the SDS-PAGE, illustrated in Figure 8, indicate that with each subsequent purification step, LDH was purified successfully. By observing the initial purification steps represented by the clarified homogenate (lane 1) and 65% cut pellet (lane 2), it is difficult to view well resolved bands indicative of successful purification. However, as the LDH progressed through the affinity (lane 3) and size exclusion (lane 4) purification steps, the bands become more tightly clustered together and resolved. Furthermore, there is significantly less streaking. Both observations led to the conclusion that LDH was successfully purified and most contaminating protein eliminated. Therefore, using SDS-PAGE to supplement the native gel electrophoresis results, it was determined that conclusion #2 was the most likely scenario. In other words, the most significant isozyme contributor was muscle type LDH with partial contribution by heart type LDH (meaning the progressively faint bands were not contaminating proteins).
The Purification Table (Table 9) is the most important figure in the Results section as it summarizes the effectiveness and utility of each purification step in the experiment. The overarching goal throughout the experiment was that with each subsequent purification step, the corresponding purification intermediate was expected to result in the loss of LDH enzyme at a lower rate compared to total protein.
Foremost, an analysis of the “Total Activity Units” column, which correlates to the amount of catalytically active LDH at each purification step, revealed that exponentially significant amounts of LDH were lost during the 65% cut pellet and affinity purified purification steps. The total LDH activity drops from 5145 U to 1170 U between clarified homogenate and 65% cut pellet, and 1170 U to 13.26 U between 65% cut pellet and affinity purified LDH. Although some LDH is expected to be lost at each step, significant amounts of LDH lost, as observed in the table and indicated above, is a point of concern. Sources of technical error during the 65% cut pellet purification step that could’ve led to significant loss of LDH were: (1) not keeping the LDH sample in ice to preserve catalytic activity; and (2) loss of sample during resuspension of pellet due to the addition of bubbles or the uptake of sample into the pipette tip. A possible source of technical error during the affinity chromatography purification step that could’ve led to significant loss of LDH was that not all of the LDH intermediate was eluted from the column. Table 3 and Figure 1 illustrate that the collection of fractions was ended significantly before the absorbance readings dropped below A(280 nm) < 0.01, since about 24 fractions were collected already. In fact, the last measured absorbance reading was 0.634. Although the absorbance values at this point of the experiment were decreasing very minimally (several hundredths at a time), an absorbance reading of this scale can indicate leftover LDH.
The “Total Protein Column”, which indicates nonessential/non-LDH protein decreases as expected and desired in an exponential fashion. This basically confirms that the experimenter has successfully removed undesired proteins from the fold. The “Specific Activity” column, which expresses the amount of LDH relative to the total protein present at each purification step, reflects a similar trend to the “Total Activity Units” column analyzed above. Essentially, it reinforces the said fact that the 65% cut pellet and affinity purification steps were unsuccessful in their purification of LDH. The 65% cut pellet expresses a specific activity (5.43 U/mg) closely similar to the clarified homogenate (5.46 U/mg), meaning that the same amount of LDH and total protein was lost, which is not desired. More significant, however, is the significant decrease in total activity between 65% cut pellet (5.43 U/mg) and the subsequent affinity purification step (1.91 U/mg). This decreased value expresses the fact that more LDH was lost relative to total protein, which is the complete opposite of the desired trend. Sources of technical error, which could’ve contributed to LDH enzyme lost is noted above. The “Fold Purification” column, which essentially quantifies how successful one was in purifying LDH, further follows the trend set by “Total Activity Units” and “Specific Activity” columns. The fold purification between clarified homogenate and 65% cut pellet were the same, indicating that the 65% cut pellet was unsuccessful in obtaining purer LDH. In other words, the purity of LDH from clarified homogenate was retained after the 65% cut pellet, rather than following an increasing trend.
Overall, the ammonium precipitation (65% cut pellet) and affinity chromatography purification steps proved to be the most unsuccessful purification steps. Significant amounts of LDH were lost between these steps as noted by the “Total Activity Units” column and more LDH was lost relative to total protein as seen in the “Specific Activity” column. Looking at the percent yield column also reinforces this notion as you go from 100% to 22.7% to 0.26% as each subsequent purification step takes place. However, the size exclusion chromatography purification step proved to be the most successful. Unlike previous purification steps where there were exponential LDH loss, there was only a slight loss of LDH activity between affinity purified (13.26 U) and size exclusion purified steps (3.65 U). Furthermore, there was an increase in specific activity which signifies that LDH was lost at an extremely slower rate compared to total protein.
One major change to the protein purification strategy that will help purify LDH is utilizing ion exchange chromatography instead of affinity chromatography. This type of column chromatography separates proteins by way of differences in their individual subunit and total protein charges (Ditlow et al, 1984). Since we know that the different isozymes of LDH exhibit characteristic isoelectric points, we can utilize a column resin composed of positively charged DEAE-cellulose and a pH buffer of 8-9 (Yin et al, 1984). This buffer will cause the LDH isozymes to become negatively charged since their isoelectric points are less than the buffer pH. Their negative charge will cause them to have a high affinity/interaction with the positively charged column resin, requiring higher concentrations of wash (i.e NaCl with phosphate buffer) to elute them. Assuming the successful use of this purification technique, one would expect values for total activity unis, which measure the amount of LDH enzyme should decrease by a smaller amount as compared to the exponential decrease observed in the affinity purification step. Furthermore, specific activity, percent yield, and fold purification values should increase as more LDH is being retained (rate of loss is lower).
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