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. Author manuscript; available in PMC: 2008 Oct 6.
Published in final edited form as: Methods Enzymol. 2004;378:31–67. doi: 10.1016/S0076-6879(04)78003-9

Table 3.

Purification Schemes for Five Recombinant Human Dihydrodiol Dehydrogenase (AKR) Isoforms a

protein purification step volume (mL) total protein (mg) total activity (:mol min-1 mg-1) specific activity (:mol min-1 mg-1) purification factor (-fold) yield (5%)
AKR1A1 sonicate 70 470 668 1.42 1 100
DE52 cellulose 12.5 88.5 233 2.63 1.85 34.7
Blue-Sepharose 7.1 30.5 183 6.0 4.23 27.4
AKR1C1 sonicate 61 460 59 0.13
DE52 cellulose 16 23 46 2.0 15 78
Blue Sepharose 8 20 42 2.1 16 71
AKR1C2 sonicate 77 510 42 0.08
DE52 cellulose 18 19 39 2.1 25 93
Blue-Sepharose 11 15 37 2.5 31 88
AKR1C3 sonicate 51 310 65 0.21
DE52 cellulose 56 25 51 2.0 10 78
Blue-Sepharose 5 12 34 2.8 13 52
AKR1C4 sonicate 44 270 4.6 0.02
DE52 cellulose 12 23 2.8 0.12 6 61
Blue-Sepharose 8 13 2.7 0.21 11 59
a

Specific activities throughout the purifications were measured using 75 :M androsterone (AKR1C4) or 1 mM 1-acenaphthenol (AKR1C1-AKR1C3) as the substrate in reaction mixtures containing 100 mM KPO4 (pH 7.0) and 2.3 mM NAD+ at 25.0 °C.