Ornithine Aminotransferase (δ-OAT) Activity Assay Kit

Product code: 55750

Method: Spectrophotometric method

Product Introduction

Proline is an important osmotic regulator that helps plants adapt to stress conditions. In higher plants, proline is synthesized through two pathways based on the initial substrate: the glutamate (Glu) pathway and the ornithine (Orn) pathway.

Ornithine aminotransferase (δ-OAT) is a key enzyme in the ornithine-based pathway for proline synthesis and plays an important role in plant stress adaptation. Ornithine and α-ketoglutarate undergo an aminotransferase reaction catalyzed by ornithine aminotransferase and NADH to generate pyrroline-5-carboxylic acid (P5C), while producing NAD. The change in absorbance at 340 nm reflects the level of ornithine aminotransferase activity.

Package Contents

Quality and Safety Information

Transportation and Storage

Instructions for Use

1. Enzyme Solution Extraction

Tissue Samples

Use a sample mass (g) to extraction solution volume (mL) ratio of 1:5-10. It is recommended to weigh approximately 0.1 g sample and add 1 mL extraction solution.

1. Add the extraction solution to the sample.
2. Homogenize in an ice bath.
3. Centrifuge at 4 °C, 10000g for 10 min.
4. Collect the supernatant and keep it on ice for testing.

Cell Samples

Use a cell count (10⁴cells) to extraction solution volume (mL) ratio of 500-1000:1. It is recommended to add 500 × 10⁴cells to 1 mL extraction solution.

1. Disrupt the cells by ultrasonic treatment in an ice bath at 300 W, with 3 seconds sonication and 7 seconds interval, for a total time of 3 min.
2. Centrifuge at 4 °C, 10000g for 10 min.
3. Collect the supernatant and keep it on ice for testing.

Liquid Samples

Test liquid samples directly.

2. Reagent Preparation

• Before use, add 20 mL Reagent I to Reagent II and dissolve thoroughly. Store unused reagent at 4 °C.
• Before use, add 20 mL Reagent I to Reagent III and dissolve completely. Store unused reagent at 4 °C.
• Before use, add 10 mL Reagent I to each bottle of Reagent IV and dissolve completely. Prepare fresh and use immediately.

3. Measurement Procedure

1. Preheat the spectrophotometer for 30 min and set the wavelength to 340 nm.
2. Preheat the prepared Reagents II, III, and IV at 37 °C for 5 min.
3. Use a 1 mL quartz cuvette. Add 300 μL Reagent II, 300 μL Reagent III, 300 μL Reagent IV, and 100 μL crude enzyme solution in sequence.
4. Mix thoroughly.
5. Record the initial absorbance at 340 nm as A1.
6. React at 37 °C for 10 min and record the absorbance as A2.
7. Calculate ΔA = A1 - A2.

Powdered reagents must be prepared by the user before use.

Activity Calculation

Calculated by Sample Protein Concentration

Unit definition: consumption of 1 nmol NADH per milligram of tissue protein per minute is defined as one unit of enzyme activity.

δ-OAT (nmol/min/mg prot) = ΔA ÷ (ε × d) × V_{total reaction}÷ (V_sample× Cpr) ÷ T = 160.77 × ΔA ÷ Cpr

Calculated by Sample Mass

Unit definition: consumption of 1 nmol NADH is defined as one enzyme activity unit.

δ-OAT (nmol/min/g fresh weight) = ΔA ÷ (ε × d) × V_{total reaction}÷ (W × V_sample÷ V_{total sample}) ÷ T = 160.77 × ΔA ÷ W

Calculated by Cell Number

Unit definition: consumption of 1 nmol NADH per 10⁴cells per minute is defined as one enzyme activity unit.

δ-OAT (nmol/min/10⁴cells) = ΔA ÷ (ε × d) × V_{total reaction}÷ (V_sample× cell number ÷ V_{total sample}) ÷ T = 160.77 × ΔA ÷ cell count

Calculated by Liquid Volume

Unit definition: consumption of 1 nmol NADH per milliliter of liquid per minute is defined as one enzyme activity unit.

δ-OAT (nmol/min/mL) = ΔA ÷ (ε × d) × V_{total reaction}÷ V_sample÷ T = 160.77 × ΔA

Formula Parameters

Precautions

• Before the formal assay, select 2-3 samples with large expected differences for a preliminary test.
• Instruments and supplies to be prepared by the user: balance, refrigerated centrifuge, mortar, UV-visible spectrophotometer, and 1 mL quartz cuvette.