Dithiothreitol (DTT) is a chemical that is often used as an antioxidizing (stron
ID: 1006869 • Letter: D
Question
Dithiothreitol (DTT) is a chemical that is often used as an antioxidizing (strongly reducing) agent. Many such agents are sulfur compounds that have a slight "rotten eggs" smell to them.
You isolated a snake venom enzyme for laboratory testing and added it to a preservative that contains DTT. To your chagrin, you find out that your enzyme has lost most of its activity after being added to the preservative. What could be the reason behind this? Choose the most likely answer.
DTT reversed the delta-Go' of the reaction that the enzyme helps to catalyze
DTT has inhibited the enzyme by reacting with an active-site serine residueExplanation / Answer
The enzyme has lost its conformation and activity due to breaking of disulfide bonds ----- correct answer
1) Proteins are soluble and most stable when folded (laws of thermodynamics, entropy gains by folding), nobody wants to purify unfolded, catalytically inactive protein. 2) DTT does not cause unfolding. Does not. Proteins are often found in highly reducing environments, so DTT stops oxidation of the protein (especially helpful in metal containing proteins) and the formation of unwanted disulfide bonds. If your protein requires disulfide bonds then you may not want to use DTT. 3) if you are doing nickel or cobalt affinity chromatography DTT is not recommended, it may alter binding. DTT also helps in preventing formation of non-specific intramolecular disulfide linkages that may alter its structure and function. DTT can also prevent unwanted binding of the target protein with contaminating Cys-rich proteins through oxidation-induced formation of intermolecular disulfide bridges. However, addition of DTT is not advisable in purifying metalloenzymes; although some metalloenzymes can still coordinate the required metal ion(s) when reconstituted back into the solution. Cytoplasmic proteins usually lack disulfide bonds. To keep the cysteine side chains in their normal reduced state, a reducing agent such as DTT is included in the purification. For proteins that have disulfide bonds as part of their native structure (such as antibodies), reducing agents may be avoided during purification to prevent disulfide reduction.