Sodium nitrite standard solution: Accurately weigh 0.1g of sodium nitrite that has been dried in a dryer for 24 hours, dissolve it in water, transfer it to a 500ml volumetric flask, dilute with water to the mark and mix well. At this time, the solution is equivalent to 200 micrograms of sodium nitrite per milliliter.
NaNO2 standard usage solution: Before use, take 5 ml of NaNO2 standard solution and place it in a 200 ml volumetric flask, dilute with water to the mark. At this time, the solution is equivalent to 5 micrograms of NaNO2 per milliliter.
Sulfanilic acid solution: Weigh 0.4g of Sulfanilic acid and dissolve it in 100 ml of 20% hydrochloric acid. Mix well and place in a brown bottle for storage in the dark.
N-(1-Naphthyl)ethylenediamine dihydrochloride solution: Weigh 0.1g of N-(1-Naphthyl)ethylenediamine dihydrochloride and dissolve it in 50 ml of water. Mix well and place it in a brown bottle for storage at 4℃, preferably used immediately after preparation.
Accurately absorb 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, and 5.0 mL of NaNO2 standard usage solution into a 50 mL volumetric flask, add 40 mL of ultrapure water, shake well, add 2 mL of Sulfanilic acid solution and shake well. React for 5 minutes in the dark; then add 1 mL of N-(1-Naphthyl)ethylenediamine dihydrochloride solution and dilute to the mark with water to make a total volume of 50 mL. Mix well and let stand for 15 minutes in the dark. Set the wavelength of the spectrophotometer to be 538 nm, respectively measure the absorbance values with NaNO2 solution concentration as the abscissa and absorbance value as the ordinate, draw the NaNO2 standard curve. NaNO2 has a good linear relationship with A538 in the concentration range of 0-0.25 μg/mL. Linear regression is performed on A538 against NaNO2 concentration to obtain the regression equation as follows: y=1.0583x+0.0015 (R2=0.99872); y represents absorbance value A538; x represents NaNO2 concentration, μg/mL.
Take a clean test tube and add glucose (1%), NaNO2 (50μg/mL) (1mL), phosphate buffer (pH7.2) (0.2 mol/L) (7mL), enzyme solution (1mL), incubate at 40℃ for 24 hours. Add Sulfanilic acid solution (0.4%) (2mL), mix well and let stand for 5 minutes in the dark; then add N-(1-Naphthyl)ethylenediamine dihydrochloride solution (0.2%) (1mL), let stand for 15 minutes and measure absorbance at a wavelength of 538 nm to calculate enzyme activity. Use phosphate buffer instead of enzyme solution as blank control, use phosphate buffer instead of NaNO2 solution and enzyme solution as zero adjustment value.
The size of enzyme activity is measured according to how much NaNO2 is degraded in the reaction system. The initial content of NaNO2 in the reaction system minus the remaining amount of NaNO2 after a period of reaction time. The more NaNO2 is degraded at the same time, the greater the activity of the enzyme. Enzyme activity calculation formula X1-X2 is the difference between NaNO2 before and after (μg/mL), t is enzyme catalysis time.
Take a clean test tube, add 1 mL of 1% glucose, 1 mL of 50 μg/mL NaNO2, 7 mL of 0.2 mol/L phosphate buffer (pH=7.2), and 1 mL of enzyme solutions. React for 1 hour in a water bath at 37°C and 50°C. Add 2 mL of 0.4% sulfanilic acid solution, mix well, avoid light and let stand for 5 min. Then add 1 mL of 0.2% N-(1-Naphthyl)ethylenediamine dihydrochloride solution, let stand for 15 min, and measure the absorbance at 538 nm to calculate the enzyme activity. Phosphate buffer was used instead of enzyme solution as a blank control, and phosphate buffer was used instead of NaNO2 solution and enzyme solution as a zero adjustment value.
The size of enzyme activity is measured by the amount of sodium nitrite degraded in the reaction system. The initial content of sodium nitrite in the reaction system minus the remaining sodium nitrite after a period of reaction time. The more sodium nitrite is degraded at the same time, the greater the enzyme activity. The thermal stability of the enzyme is characterized by comparing the enzymatic activity at 37°C and 50°C.