ultimately denatures at about 90 °C it has the added ability to accurately reform its bonds and return to its original structure and function once cooled back down. Alkaline phosphatase in E. coli is located in the periplasmic space and can thus be released using techniques that weaken the cell wall and release the protein. Due to the location of the enzyme, and the protein layout of the enzyme, the enzyme is in solution with a smaller amount of proteins than there are in another portion of the cell. The proteins' heat stability can also be taken advantage of when isolating this enzyme (through heat denaturation). In addition, alkaline phosphatase can be assayed using p-Nitrophenyl phosphate. A reaction where alkaline phosphatase dephosphorylates the non-specific substrate, p-Nitrophenyl phosphate in order to produce p-Nitrophenol(PNP) and inorganic phosphate. PNP's yellow color, and its λmax at 410 allows spectrophotometry to determine important information about enzymatic activity. Some complexities of bacterial regulation and metabolism suggest that other, more subtle, purposes for the enzyme may also play a role for the cell. In the laboratory, however, mutant Escherichia coli lacking alkaline phosphatase survive quite well, as do mutants unable to shut off alkaline phosphatase production. The optimal pH for the activity of the E. coli enzyme is 8.0 while the bovine enzyme optimum pH is slightly higher at 8.5. Alkaline phosphatase accounts for 6% of all proteins in derepressed cells. Intragenic complementation When multiple copies of a polypeptide encoded by a gene form an aggregate, this protein structure is referred to as a multimer. When a multimer is formed from polypeptides produced by two different mutant alleles of a particular gene, the mixed multimer may exhibit greater functional activity than the unmixed multimers formed by each of the mutants alone. In such a case, the phenomenon is referred to as intragenic complementation. E. coli alkaline phosphatase, a dimer enzyme, exhibits intragenic complementation. When particular mutant versions of alkaline phosphatase were combined, the heterodimeric enzymes formed as a result exhibited a higher level of activity than would be expected based on the relative activities of the parental enzymes. These findings indicated that the dimer structure of E.coli alkaline phosphatase allows cooperative interactions between the constituent monomers that can generate a more functional form of the holoenzyme. Use in research By changing the amino acids of the wild-type alkaline phosphatase enzyme produced by Escherichia coli, a mutant alkaline