Description
Glucokinase (GK, EC 2.7.1.2) is a member of the hexokinase family, primarily found in mature hepatocytes and pancreatic islet cells. Under normal physiological conditions, the main role of GK is to monitor blood glucose levels. Glucokinase (GK) phosphorylates glucose to produce glucose-6-phosphate. This product is further coupled with glucose-6-phosphate dehydrogenase and NADP+, and the increase in NADPH absorbance is measured at 340 nm, thereby calculating the enzyme activity.Component50TStorageExtraction Buffer60 mL2-8℃Reagent 140 mL2-8℃Reagent 21EA-20℃Reagent 31EA2-8℃Reagent PreparationReagent 2 (Powder, 1 vial):Before opening, ensure the powder is at the bottom of the vial (tap manually if needed).Add 2.2 mL of distilled water to dissolve.The storage period is the same as the kit's expiry date.Reagent 3 (Powder, 1 vial):Before opening, ensure the powder is at the bottom of the vial (tap manually if needed).Add 36 mL of Reagent 1 to dissolve.The storage period is the same as the kit's expiry date.User-Prepared Instruments & MaterialsMortar (homogenizer), ice bucket (ice maker), benchtop centrifuge, adjustable pipettes, water bath (oven, incubator, metal bath), 1 ml quartz cuvette, centrifuge tubes, UV spectrophotometer, distilled water (deionized water or ultrapure water is acceptable).Sample Extraction1. Tissue Samples: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 12000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.Note: If increasing the sample amount, use a ratio of 1:5 to 1:10 (tissue weight (g) : Extraction Buffer volume (mL)) for extraction.2. Bacterial/Cell Samples: Collect bacteria or cells into a centrifuge tube by centrifugation and discard the supernatant. Take 5 million bacteria or cells, add 1 mL of Extraction Buffer, and disrupt using ultrasound on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 10 s, repeat 30 times). Centrifuge at 12000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.Note: If increasing the sample amount, use a ratio of 500-1000 (x10⁴ cells) : 1 (mL Extraction Buffer) for extraction.3. Liquid Samples: Detect directly. If the sample is turbid, centrifuge and use the supernatant for detection.Assay Procedure1. Preheat the UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.2. Pre-warm the prepared Reagent 2 and Reagent 3 at 25°C for 5 minutes to reach room temperature.3. In a 1 mL quartz cuvette (1 cm light path), add sequentially:Reagent (µL)Test CuvetteSample80Reagent 240Reagent 36804. Mix well. Read the absorbance at 340 nm at 1 minute (A1) and then again at 21 minutes (i.e., after 20 minutes) (A2). Calculate ΔA = A2 - A1.Notes:1. If ΔA is close to zero, the reaction time can be appropriately extended to 30 minutes or longer before reading A2. The modified reaction time (T) must be substituted into the calculation formula. Alternatively, increase the sample volume appropriately. The modified sample volume (V1) must be substituted into the calculation formula.2. If the increasing trend is unstable, read the absorbance every 10 seconds and select a linear increasing period for calculation. The corresponding ΔA value should be substituted into the calculation formula.GK Activity Calculation1. Based on Sample Protein Concentration:Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per mg of protein.Formula:GK (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (V1 × Cpr) ÷ T = 80.4 × ΔA ÷ Cpr2. Based on Sample Fresh Weight:Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per gram of fresh tissue.Formula:GK (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (W × V1 ÷ V) ÷ T = 80.4 × ΔA ÷ W3. Based on Bacterial/Cell Density:Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per 10⁴ bacteria/cells.Formula:GK (nmol/min/10⁴ cell) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (500 × V1 ÷ V) ÷ T = 0.16 × ΔA4. Based on Liquid Volume:Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADPH per minute per mL of liquid.Formula:GK (nmol/min/mL) = [ΔA × V2 ÷ (ε × d) × 10⁹] ÷ V1 ÷ T = 80.4 × ΔAParameter Description:ε: NADPH molar extinction coefficient, 6.22 × 10³ L/mol/cmd: Light path of the 1 mL quartz cuvette, 1 cmV: Volume of Extraction Buffer added, 1 mLV1: Volume of sample supernatant added, 0.08 mLV2: Total reaction volume, 0.8 mL = 8 × 10⁻⁴ LT: Reaction time, 20 minW: Sample mass, g500: Bacteria or cell number, in units of 10⁴Cpr: Protein concentration of the supernatant, mg/mL; Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.PrecautionsIt is recommended to first select 1-3 samples with significant differences (e.g., different types or groups) for preliminary experiments to familiarize yourself with the procedure. Determine or adjust the sample concentration based on the preliminary results to prevent unnecessary waste of samples or reagents