3-α-Hydroxysteroid Dehydrogenase

Product Overview

Total bile acid (TBA) in serum is the gold standard for assessing hepatocellular injury and cholestasis. The TBA assay kit employs 3α-HSD as its sole specific catalytic enzyme. The principle of the cyclic enzymatic assay for TBA is that 3α-HSD catalyzes the conversion of bile acids and Thio-NAD+ into 3-keto steroids and Thio-NADH, which exhibits a characteristic absorption peak at 405nm. Subsequently, 3α-HSD further catalyzes the reverse reduction of excess NADH, regenerating bile acids and amplifying the OD405signal in a cyclical manner. Clinically, this method is suitable for screening viral hepatitis, liver cirrhosis, drug‑induced liver injury, obstructive jaundice, and intrahepatic cholestasis of pregnancy (ICP). Moreover, 3α-HSD specifically recognizes α-hydroxylated steroids at the C3 position and is also used for the quantitative analysis of endocrine steroid hormones--such as androgens and etiocholanolone--enabling the screening of total androgen metabolites in serum and urine, thereby supporting the evaluation of polycystic ovary syndrome (PCOS), adrenal hyperplasia, and testicular function. Based on practical needs arising from the development and clinical application of TBA reagents, Hzymes has engineered a recombinant 3α-HSD (HH1601) through gene mining, optimization of production processes, and rigorous empirical testing. All core performance parameters have been validated, with results comparable to those of leading international competitors. This enzyme exhibits high substrate affinity, selectively recognizing only 3α-OH groups while remaining insensitive to 3β-hydroxylated steroids, thus avoiding cross-substrate interference. The protein specific activity and lyophilized powder specific activity of 3α-HSD (HH1601) are both high and stable; it operates optimally at pH8.5-9.5 and remains stable over a broad pH range of 6.0-9.0. It also demonstrates excellent thermal stability, with a long half-life at 50°C, making it compatible with mainstream one-step and two-step TBA assay systems currently available. Hzymes'TBA reagent offers an in-vessel open-bottle stability of up to 28 days, and its accelerated stability at 37°C significantly exceeds that of other brands. It features a wide linear range and robust resistance to common interfering substances in serum, such as chylomicrons, hemoglobin, and bilirubin, allowing reliable detection even with minute volumes of serum or plasma samples. 3α-HSD (HH1601) has been scaled up for industrial production, with a robust manufacturing process ensuring a protein purity exceeding 90% and extremely low levels of contaminating enzymes, thereby guaranteeing superior reagent performance. It meets global demand for mass-produced TBA reagents and complies with IVD raw material and reagent registration requirements.

Principle of the cyclic enzymatic assay for TBA

Key Features

High substrate affinity

recognizing only 3α-OH and not 3βhydroxylated steroids, with no cross-substrate interference.

High specific activity

both as a protein and in lyophilized form, along with excellent stability; it operates optimally at pH 8.5-9.5 and remains stable over a broad pH range of 6.0-9.0, rivaling leading international brands.

Superior thermal stability

with a long half-life at 50°C, providing a robust foundation for the openvial stability, accelerated stability, and longterm stability of TBA reagents.

3a-HSD	Hzymes	Vendor A	Vendor B	Vendor C	Vendor D	Vendor E	Vendor F	Vendor G
Specific activity(U/mg)	322	337	302	330	387	611	477	300
pH stability (25°C, 24hr)	5.5-9.5	5.5-9.5	5.5-9.5	5.5-9.5	5.5-9.5	5.5-9.5	5.5-9.5	5.5-9.5
Half-life at 50°C(min)	>200	162	77	>200	21	>200	Rapid inactivation	148
Reagenthalf-life at 37°C(Days)	62	44	21	52	31	60	-	17

Applications

Core application: Liver function-TBA assay.

3α-HSD (HH1601) is compatible with ThioNAD (HH3801) and NADH (HH3201), supporting the development and large-scale preparation of total bile acid (TBA) assay reagents based on a cyclic enzymatic method. It is also suitable for POCT and rapid emergency TBA dry test strips.

Quantitative analysis of endocrine steroid hormones

(such as androstenedione and etiocholanolone), for screening total androgen metabolites in serum and urine, and for assisting in the evaluation of polycystic ovary syndrome (PCOS), adrenal hyperplasia, and testicular function.

Reaction Principle

The amount of NADH can be measured at 340nm. One unit of enzyme activity is defined as the amount of enzyme required to catalyze the production of 1μmol of NADH per minute under conditions of 25°C and pH8.9.

Specification

CAS	9028-56-2
EC Number	EC 1.1.1.50
Appearance	White amorphous lyophilized powder
Dry powder specific activity	≥55.0U/mg
Protein Purity	≥90%(SDS-PAGE)
Protectants	BSA
Contaminants	Glucose-6-phosphate dehydrogenase≤0.01%; Lactate dehydrogenase≤0.005%; Glutamate dehydrogenase≤0.001%; Malate dehydrogenase≤0.01%; Hexokinase≤0.01%
Optimal pH	pH8.5-9.5
pH stability	pH6.0-9.0 (25°C, 16 h)
Optimal temperature	55°C
Km	3.6×10^-5M (Androsterone), 4.7×10^-5M (NAD+)
Temperature stability	Below 37°C (pH9.0, 30 min)
Long-term stability	Stored at -20°C for 12 months, maintaining over 90% activity.
MW	30kDa(SDS-PAGE)
pI	5.2
Inhibitors	Fe³⁺, Co²⁺, Cu²⁺, Mn²⁺, Zn²⁺

Available Formats

• HH1601-01 3kU

• HH1601-02 100kU

• HH1601-03 500kU

• HH1601-04 1000kU

• HH1601-99 Customizable

Performance Data

Excellent analytical linearity

Within the total bile acid concentration range of 0-200μmol/L, the TBA assay kit exhibits excellent analytical linearity

Good correlation with TBA reagents

The TBA reagent from Hzymes demonstrates good correlation with TBA reagents from international brands in clinical sample measurements, exhibiting both methodological robustness and measurement accuracy.

Excellent stability

3a-HSD (HH1601) confers excellent stability to the TBA reagent; when stored sealed at 4°C, it remains stable for one year, and once opened, it can be kept stable for 28 days under in-instrument conditions.