The above illustrates the alignment sequence generated using GenBank, of pig hea

Question

The above illustrates the alignment sequence generated using GenBank, of pig heart LDH (Query 1) and pig muscle LDH (subject 1). Considering the differing amino acids between the two sequences, at a pH of 8.6 the pig heart LDH is supposed to be more negatively charged and thus migrate further during gel electrophoresis (you may assume the pkas of the side chains are the same as those for the free amino acids). In query 1, for the relevant substitutions, I am seeing 2 glutamate (-1 x 2) and 1 lysine (+1) that would give a -1 charge at a pH of 8.6. For subject 1, I see 1 aspartate (-1), 3 histidines (-1 x3), 1 lyisne (+1) and 1 glutamate (-1) that would give a -4 charge at the same pH. What am I doing wrong?

Explanation / Answer

Here, histidine should not be counted as negatively or positively charged since its side chain has pKa of 6.0 only which makes it neutral at higher pH. The calculared overall theoretical pI of querry 1 and subject 1 proteins are 4.65 and 5.42 respectively. This explains the more negative behaviour of querry 1 at pH 8.6 because for higher pH, the overall charge will be more negative.

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