TABLE 3.

Purification of the PDH complex of Z. mobilis

Step	Total activity (U)	Total protein (mg)	PDH complex sp acta (U/mg)	Purification (fold)
Cell extract	155	15,500	0.01	1
Protamine sulfate	150	15,030	0.01	1
6% PEG	220	7,350	0.03	3
10% PEG, 0.75 mM MgCl2	159	1,980	0.08	8
Anion-exchange column	62	60	1.04	104
Gel filtration	13.9	1.1	12.6	1,260

^aPDH complex activity was measured in cell extract and after the first two purification steps by the reduction of ferricyanide at 430 nm, because direct measurement of NADH formation by the PDH complex at 340 nm was obscured due to the reaction of pyruvate decarboxylase, which caused a rapid decrease in pyruvate concentration in the test mixture and a concomitant decrease in extinction (the ɛ₃₄₀ of pyruvate is 0.023 mM⁻¹ cm⁻¹). The enzyme activities at further purification steps were determined by the formation of NADH.