Experiments in Biochemistry
Notes 1. Arrive the lab on time. Preview the content of the experiment. 2. During the experiment, no unnecessary noise. 3. Pay attention to the safety of the experiment. Wear a lab coat. In case of danger, immediately cut off water, electricity, and fire, and report to the teacher. 4. Experiments are performed in groups, each contains 2 students. 5. Take care of your lab items. Do not turn on unused instruments. 6. Listen carefully before you begin. If you encounter unclear experimental steps, consult the teacher in time and carefully record the various phenomena and experimental results in the experiment. 7. After the experiment, be sure to wash and tidy up the glassware and instruments you have used. Your results of the experiment need to be approved by the teacher. Get permission before leaving. 8. Arrange the day students to clean the laboratory. 9. Submit the experiment E-report on time
Experiment 1 The main chemical properties of amino acids and proteins 1. Objective To experimentally verify the main chemical properties of amino acids and proteins, and enhance perceptual understanding. 2. Principles Both α-amino acids and proteins contain the α-amino acyl group structure, which allows them to react with ninhydrin in the presence of water to produce a blue-purple substance. [nɪnˈhaɪdrɪn]
2.1 The ninhydrin reaction Note: Proline and hydroxyproline react with ninhydrin to produce a (bright) yellow substance. NOT purple
Ninhydrin test principle The amino group belonging to a free amino acid undergoes a chemical reaction with ninhydrin, which behaves as an oxidizing agent. When exposed to ninhydrin, the amino acid undergoes oxidative deamination, resulting in the liberation of CO2 , NH3 , and an aldehyde along with hydrindantin (which is a reduced form of ninhydrin). Now, the ammonia goes on to react with another ninhydrin molecule to form diketohydrin (which is also known as ruhemann’s complex). This complex is responsible for the deep blue color. When the analyte contains imino-acids like proline, a yellow colored complex is formed. When asparagine is used, the color of the resulting complex is brown
2.2 The biuret test [bjə'ret]
Biuret test is used to detect the presence of peptide bonds in the sample and to test for the presence of proteins or peptides. Proteins and peptides are polymers of amino acids. They are chains of amino acids, which are covalently bound to each other by a covalent bond, called a peptide bond, between the carbon number one (C1 ) of one amino acid and nitrogen number two (N2 ) of adjacent amino acid. The formation of a peptide bond is a condensation reaction. In an alkaline environment, in the presence of the cupric ion (Cu+2) and in the biuret reagent, the peptide bond can be used to form a violet or purple-colored complex. [ˈkjuːprɪk]
2.3 Identification of S-containing amino acids If a protein molecule contains sulfur-containing amino acids such as cysteine and methionine, upon heating with an alkali, it will decompose to produce sulfide ions. These sulfide ions, when encountering lead salts, will form a black precipitate of lead sulfide
Protein colloids are hydrophilic colloids. If the hydration film and charge on their colloidal particles are removed, the protein colloidal particles will aggregate and precipitate. This is known as the precipitation of proteins. There are various methods to precipitate protein colloids, with the following being commonly used: 2.4 Aggregation and precipitation of proteins