Polypeptide research Urea denaturates proteins

Core tips:

Key words: urea denatured protein

Key words: denaturation of protein denaturation of protein

1、 Purpose of the experiment

1. Understand the meaning of protein denaturation.
2. Master the types of common protein denaturants.

2、 Experimental principle

The peptide chains in the natural egg self-made molecules twists and turns in a certain way to form a specific conformation. The maintenance of this conformation mainly depends on the hydrogen bond in the protein molecule. Urea can destroy the hydrogen bond, lead to the relaxation of protein molecular structure, denaturation of protein, after denaturation, the peptide chain of protein will be extended, so that the original sh contained in the molecule can be exposed, and it can interact with mercapto reagent, in a certain range, the exposed sh will increase with the deepening of the degree of denaturation, so the determination of SH The increase in egg quality can be measured by the degree of denaturation. In alkaline condition, – SH can be oxidized by n [Fe (no) (CN) 5] 2-nitroso-ferricyanide to – S-S (disulfide bond). The iron of oxidant is reduced from high price to low price, and its complex is red.

The amount of red complex produced is directly proportional to the amount of – SH, which can be determined by Colorimetry (520nm). Because – SH is easily oxidized by air oxygen under the catalysis of heavy metal ions, a little oxide is added to the reaction system to inhibit this reaction.

3、 Instruments, raw materials and reagents

instrument

Tube 1.5cm × 15cm (× 27), calibration tube 5.0m1 (× 18), pipette 0.10ml (× L), 0.50ml (× 4), l.0ml (× 2)

5.0m1 (× L), electronic balance, 722 (or 7220) spectrophotometer, volumetric flask 10ml (× L).

raw material

Ovalbumin or other protein samples

reagent

1. 2% ovalbumin solution: weigh 0.5g of ovalbumin and dissolve it in distilled water ① (in order to reduce protein denaturation, only use glass rod to gently stir the solution). ）Remove the Insolubles by centrifugation and dilute the supernatant with water to 25ml.
2. Urea (A.R.): in powder form.
3. Cys HCl: accurately weigh 15.76mg of cysteine hydrochloride into a 10.0ml volumetric flask, dilute to the scale with distilled water, and shake well. When using, take 0.5ml of this solution and dilute it to 10.0ml with distilled water. Each ml of this diluent contains 0.5 μ mol of Cys HCl.
4. Saturated sodium chloride (A.R.) solution
5. 0.067mol/lnacn-1.5mol/lna2co3 solution: weigh 3.28g NaCN (A.R.) and 159G nna2co3 (A.R.), dissolve in distilled water and dilute to 1000ml.
6. 2% sodium nitroferricyanide solution: weigh 2G of sodium nitroferricyanide (A.R.), dissolve in distilled water and dilute to 100ml.

4、 Operation steps

（1） – drawing of SH standard curve

Take 9 tubes, number them according to the table below and add reagents.

Shake well to compare colors. The color is stable within 15s and the operation is fast. It is not allowed to wait for each tube to be added with developer before testing. One tube shall be added for testing.

Calculate the number of – SH in each tube according to the concentration of semihazy ammonia hydrochloride solution and the amount added in each tube, and fill in the table with this number and the measured absorbance value. The standard curve is drawn with the number of – SH as abscissa and the absorbance value as ordinate.

（2） Determination of denaturation degree of protein (expressed by the measured amount of – SH)

Take 18 5.0ml calibration tubes, control and test two groups respectively. The control group is numbered 0-8, and the test group is numbered 0 ˊ – 8 ˊ. Add 0, 0.8, 1.0, l.2, 1.4, 1.6, 1.8, 2.0 and 2.2g urea in the control group

A little distilled water, after urea dissolves, add water to the scale. Urea 0, 0.8, 1.0, l.2, 1.4, 1.6, 1.8, 2.0 and 2.2g were successively added into the calibration tube 0 ˊ to 8 ˊ of the determination group.

Add 2.0ml of ovalbumin solution, dissolve urea, place for 45min, and add water to the scale. Another 18 tubes were taken and divided into two groups: control group and test group. The number of control group and test group were 0-8 and 0 ˊ – 8 ˊ respectively. 3.0ml saturated NaCl solution and 0.067mol/lnacn ~ 1.5mol/lna2co3 solution were added to each tube.

Take 1.0ml solution from each of the first 18 calibration tubes, respectively, and add 0.5ml of 2% nitrosylferricyanide solution to each tube. Shake the Colorimetry (520nm) immediately, and the operation time shall not exceed 15s. When comparing colors, use the zero tube of this group to adjust zero.

Subtract the absorbance value of the corresponding number of tubes in the control group from the absorbance value of each tube in the measurement group 0 ˊ ~ 8 ˊ. One sh number of each tube is obtained by comparing with the standard curve. Taking the number of SH as the ordinate and the amount of urea as the abscissa, the graph is drawn and the results are explained.

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