c e n z enzymes h y cholesterol e m dehydrogenase se

O R I G I N Nocardia sp.

� S P E C I F I C A T I O N SAppearance Freeze dried powderActivity ≥5 U/mg powder

� A P P L I C A T I O NUsed in the formulation of Cholesterol testing reagents or in biosensor applications.

� U N I T D E F I N I T I O NOne unit of activity is defined as the amount of enzyme that will catalyse the productionof 1.0 micromole of NADH per minute at 25°C under standard assay method conditions.

CholesterolDehydrogenase

E N Z Y M E S

CAT# C

HDE-70-1241

C A T # C H D E - 7 0 - 1 2 4 1

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� A S S AY P R I N C I P L ECholesterol Dehydrogenase (CDH) catalyses the following reaction:

Cholesterol + NAD+ Cholest-4-en-3-one + NADH + H+

The appearance of NADH is measured at 340nm by spectrophotometry.

CDH

Molecular Weight: 37kDa (SDS-PAGE)

Isoelectric Point: 4.5

Kmvalues: Cholesterol; 1.5 x 10-4 MNAD+; 2.3 x 10-4 M

Optimum pH (Fig. 1): pH 10.0

Optimum Temperature (Fig. 2): 30°C

pH Stability (Fig. 3): 6.5 to 7.5 (37°C for 15 minutes)

Thermal Stability (Fig. 4): Stable at 35°C and below (pH 7.0 for 5 minutes)

Inhibitor: Ag+

Activator: Triton X-100

� C H A R A C T E R I S T I C S

CAT# C

HDE-70-1241

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SRELATIVE ACTIVITY (%)

pH

120

100

80

60

40

20

0

7 8 9 10 11

F IGURE 1 : OP T IMUM pH

F IGURE 4 : THERMAL S TAB I L I TY(pH 7 . 0 FOR 15 M INS . )

F IGURE 2 : OP T IMUM T EMPERATURE

F IGURE 3 : pH S TAB I L I TY ( 37 °C FOR 15 M INS . )

pH

6 7 8 9 10 11

RELATIVE ACTIVITY (%)

TEMPERATURE (°C)

TEMPERATURE (°C)

120

100

80

60

40

20

0

20 30 40 50 60

RESIDUAL ACTIVITY (%)

120

100

80

60

40

20

0

RESIDUAL ACTIVITY (%)

120

100

80

60

40

20

0

10 20 30 40 50 60 70

SUBSTRATE RELATIVE ACTIVITY (%)

Cholesterol 100%

β-Sitosterol 52%

Ergosterol 50%

Stigmasterol 30%

Pregnenolone 14%

Lanosterol 0%

SUBSTRATE RELATIVE ACTIVITY (%)

Dehydroisoandrosterone 0%

Cholic acid 0%

Testosterone 0%

NAD 100%

NADP 0%

TABLE 1 : SUBS TRATE SPEC I F IC I TY OF CHOLES T EROL DEHYDROGENASE

CHEMICAL CONCENTRATION RELATIVE ACTIVITY(mM) (%)

None — 100%

MnCl2 1.0 52%

MgCl2 1.0 50%

CaCl2 1.0 14%

FeCl2 1.0 0%

CHEMICAL CONCENTRATION RELATIVE ACTIVITY(mM) (%)

LiCl2 1.0 96%

NiCl2 1.0 96%

ZnCl2 1.0 48%

AgNO3 1.0 0%

TABLE 2 : E F F ECT OF VAR IOUS CHEMICALS ON CHOLES T EROL DEHYDROGENASE

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CAT# C

HDE-70-1241

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