3–HYDROXYBUTYRATE DEHYDROGENASE [3–HBDHⅡ] (T-68)

(Diagnostic Reagent Grade) Nagase Diagnostics ENZYMES T-68REACH適合品

3–HYDROXYBUTYRATE DEHYDROGENASE [3–HBDHⅡ]

from Alcaligenes faecalis
(D–3–Hydroxybutyrate: NAD+ oxidoreductase, EC 1.1.1.30)

D–3–Hydroxybutyrate + NAD+ → Acetoacetate + NADH + H+

Preparation and Specification

Appearance
: White amorphous powder, lyophilized
Specific activity
: More than 1,500 U/mg solid

Properties

Substrate specificity
: See Table 1
Molecular weight
: 60±5 kDa (TSK G–3000SW)
30±5 kDa (SDS–PAGE)
Isoelectric point
: pH 5.0±0.2
Michaelis constants
: D–3–Hydroxybutyrate 1.6 × 10-3M
Optimum pH
: 8.5Figure 1
pH stability
: 5.5–11.0 (37℃, 60 min) Figure 2
Optimum temperature
: 45℃ (Tris–HCl buffer) Figure 3
Thermal stability
: Stable at 37℃ and below (pH 8.5, 10 min) Figure 4
Effect of metal ions
: See Table 2
Effect of detergents
: See Table 3

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of ketone bodies when coupled with acetoacetate decarboxylase (AADC) , thio–NAD and NADH.

Table 1. Substrate specificity

SubstrateRelative activity
(%)
3–Hydroxybutyric acid100
2–Hydroxybutyric acid0
D,L–Lactic acid0
D,L–Malic acid0
Gluconic acid0
Glycolic acid0

 

Table 2. Effect of metal ions on 3–HBDH Ⅱ activity

SubstrateRelative activity
(%)
None100
LiCl104
NaCl101
NH4Cl101
KCl98
CsCl100
CuCl213
BaCl2107
ZnCl288
PbCl260
NiCl249
CoCl244
MnCl240
CaCl291
MgCl294
FeSO491
FeCl3103
EDTA85
NaN3102

Table 3. Effect of detergents on 3–HBDH Ⅱ activity

Detergent (0.1%)Relative activity
(%)
None100
PluronicL–7157.3
 P–10394.7
 F–6868.7
AdekatolSO–120110
 LO–7109
 NP–690112
 PC–893.9
 NP–72054.2
NikkolSL–1062.9
 TL–1074
 MGO55.7
 TMGO554.2
 MYO–675.6
 MYL–1032.8
 BL–20TX101
 NP–18TX99.2
 OP–10104
 HCD–10091.6
TX–100100
Tween 80 65.6

Fig.1 pH Optimum


〇: Acetate buffer
●: Phosphate buffer
□: Tris-HCI buffer
■: Glycine-NaOH buffer

Fig.2 pH Stability


37℃, 60min
〇: Citrate buffer
●: Acetate buffer
□: Phosphate buffer
■: Tris-HCI buffer
△: Glycine-NaOH buffer

Fig.3 Optimum Temperature


pH 8.5
50 mM Tris-HCI buffer

Fig.4 Thermal Stability


pH 8.5, 10min.
50 mM Tris-HCI buffer

Assay

Principle
  1. The assay is based on the increase in absorbance at 340 nm as the formation of NADH proceeds in the following reaction:

3–HBDH Ⅱ
3–Hydroxybutyrate+NAD+Acetoacetate+NADH+H+

NAD:Nicotineamido adenine dinucleotide
Unit definition
  1. One unit is defined as the amount of enzyme which converts 1 μ mole of 3–Hydroxybutylate to acetoacetate per minute at 37℃ under the conditions specified in the assay procedure.

Reagents
  1. Reaction mixture
    Dissolve 126 mg of D– (–) –3–hydroxybutylic acid with 12.5 ml of 0.2 M Tris–HCl buffer pH 8.5 and add 25 ml of distilled water and 12.5 ml of 10 mM NAD solution.
  2. Enzyme dilution buffer
    20 mM Tris–HCl buffer pH 8.5 containing 0.1% (W/V) BSA.
  3. Reagents
    NAD: NACALAI TESQUE, INC. #24334–84
    D–(–)–3–Hydroxybutylic acid (Na salt) :
    Sigma Chemical Co. #29836-0
    BSA: Millipore Fraction V pH5.2 #81–053
Enzyme solution
  1. Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it
  1. with enzyme dilution buffer to adjust the concentration as required.
Procedure
  1. Pipette accurately 3.0 ml of reaction mixture into a small test tube and preincubate at 37℃.
  2. After 5 min, add exactly 40 μl of enzyme solution and mix to start the reaction at 37℃.
    In the case of a test blank, add 40 μl of enzyme dilution buffer in place of enzyme solution.
  3. After starting the reaction, measure the rate of increase per minute in absorbance at 340 nm. The rate must be measured within the linear portion of the absorbance curve.
    Absorbance sample :As/min
    blank :Ab/min
    △A/min = (As/min-Ab/min) ≦ 0.070 Abs/min
Calculation
  1. Activity (U/mg of powder) = {(△A/min)/6.22} × 3.04/0.04 × 1/x
    6.22:millimolar extinction coefficient of NADH at 340 nm
    (cm2 /μmole)
    3.04 :final volume (ml)
    0.04 :volume of enzyme solution (ml)
    X :concentration of the sample in enzyme solution
    ( mg/ml)
Storage
  1. Storage at -20℃ in the presence of a desiccant is recommended.

3–HBDH Ⅱ活性測定法 (Japanese)

試薬液
  1. 反応試薬混合液
    3–ヒドロキシ酪酸126mg を0.2M トリス–HCl 緩衝液pH8.5 12.5ml で溶解した後、精製水25ml と10mM NAD 溶液12.5ml を混合する。
  2. 酵素溶解希釈用液
    0.1% (W/V) BSA を含む20mM トリス–HCl 緩衝液pH8.5
  3. 試薬
    NAD (ニコチンアミドアデニンジヌクレオチド)
    :ナカライテスク製 #24334–84
    3– ヒドロキシ酪酸[D– (–) –3– ヒドロキシ酪酸・ナトリウム塩]:シグマ製 #29836-0
    BSA: Millipore 製 Fraction V pH5.2 #81–053
酵素試料液
  1. 検品約20mg を精密に量り、酵素溶解希釈用液に溶解して全容20ml とする。
    その液を酵素溶解希釈用液で適宜希釈する。
測定操作法
  1. 小試験管に反応試薬混合液3.0ml を正確に分注して37℃で予備加温する。
  2. 5 分経過後、酵素試料液40 μl を加えて混和し、37℃で反応を開始する。
    盲検は酵素試料液の代わりに酵素溶解希釈用液40 μl を加える。
  3. 反応開始後、340nm における吸光度を測定して直線的に反応している1 分間当たりの吸光度変化を求める。
    求められた吸光度変化を試料液はAs/min、盲検液はAb/min とする。
    ΔA/min = (As/min-Ab/min) ≦ 0.070 Abs /min
計算
活性 (U/mg) = {(ΔA/min)/6.22} × 3.04/0.04 × 1/x
6.22 :NADH の340nm におけるミリモル分子吸光係数
(cm2 /μmole)
3.04 :反応総液量 (ml)
0.04 :反応に供した酵素試料液量 (ml)
X :酵素試料液中の検品濃度 (mg/ml)