Chemistry 1031 North Park University The Isolation of the Protein Casein Introduction Casein is the most important protein in milk. Casein can be isolated from milk by acidification to bring it to its isoelectric point. Proteins are least soluble in water at their isoelectric point (pI) because the number of positive charges on a protein equals the number of negative charges. The positive end of one protein molecule attracts the negative end of another protein molecule, and the proteins aggregate together and the aggregates precipitate out of solution. When the pH = pI, the net molecular charge is zero and consequently there are no charge-repulsive forces that promote solubility. On the other hand, if the protein molecule has a net positive charge (at pH < pI) or a net negative charge (at pH > pI), its solubility in water is increased because of charge-repulsion between the identically charged molecules. In the first part of this experiment, you will isolate casein from milk (pH is about 7) by utilizing its characteristic isoelectric point (pH = 4.6). Casein will be separated as an insoluble precipitate by acidification of the milk to its pH at 4.6. If whole milk is used, the fat that precipitates along with casein can be removed by dissolving the fat in alcohol and removing the insoluble protein by filtration. We will use skim milk with very little fat to avoid this step. In the second part of this experiment, you will demonstrate that the precipitated milk product is a protein. The identification will be achieved by performing two important chemical tests. A. BIURET TEST This is one of the most general tests for proteins. When a protein reacts with copper(II) sulfate in the Buiret Reagent, a positive test is the formation of a copper complex that has a pink-blue to violet color. B. NINHYDRIN TEST Amino acids with a free –NH2 group and proteins containing free amino groups react with ninhydrin to give a purple-blue complex. Chemistry 1031 North Park University Procedure Part I: 1. To a 125-mL Erlenmeyer ﬂask, add 20 mL of skim milk and heat the ﬂask in a water bath (a 600-mL beaker containing about 200 mL of tap water). Secure the flask with a ring stand (Figure 1). Stir the milk solution constantly with a stirring rod. Use a Lab Quest with a temperature probe to monitor the temperature. When the bath temperature has reached about 40°C, remove the ﬂask from the water bath, and add about 5 drops of glacial acetic acid while stirring (1-2 min). Allow contents to settle. Observe the formation of the precipitate of casein. Figure 1: Experimental set up for precipitation of casein. 2. Filter the mixture into a 100-mL beaker by pouring it through 1 coffee filter (make a small well in the filter before pouring) which is fastened with a rubber band over the mouth of the beaker (Figure 2). Remove most of the water from the precipitate by squeezing the filter gently. Discard the ﬁltrate in the beaker into a waste beaker in the hood. Using a spatula, scrape the precipitate from the coffee filter onto a piece of pre-weighed filter paper. Figure 2: Filtration of casein. Chemistry 1031 North Park University 3. Spread the casein on a paper towel and let it dry. Determine the mass of the dried casein and calculate the percentage of casein in the milk. Record it on your Report Sheet. % 𝐶𝑎𝑠𝑒𝑖𝑛 = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑖𝑑 (𝑐𝑎𝑠𝑒𝑖𝑛) 𝑥 100 20.0 𝑚𝐿 𝑜𝑓 𝑚𝑖𝑙𝑘 Part II: Biuret Test 1. Place 15 drops of each of the following solutions in ﬁve clean, labeled test tubes. a. 2% glycine b. 2% gelatin c. 2% albumin d. Casein prepared above in Isolation of Casein (pea-sized amount of casein) + 15 drops of distilled water e. 1% tyrosine 2. To each of the test tubes, add 5 drops of Biuret solution while swirling. The development of a purplish-violet color is evidence of the presence of proteins. Record your results on the Report Sheet. Ninhydrin Test 1. Place 15 drops of each of the following solutions in ﬁve clean, labeled test tubes. a. 2% glycine b. 2% gelatin c. 2% albumin d. Casein prepared above in Isolation of Casein (pea-sized amount of casein) + 15 drops of distilled water e. 1% tyrosine 2. To each of the test tubes, add 3 drops of ninhydrin reagent and heat the test tubes in a boilingwater bath for about 5 min. Amino acids with a free –NH2 group and proteins containing free amino groups react with ninhydrin to give a purple-blue complex. Record your results on the Report Sheet. Chemistry 1031 North Park University The Isolation of the Protein Casein Name:______Lydia Thomas ___________________________________________________ Lab Partner(s):_Jed , Annok, Carina, Adrianna ________________________________________ Date:________12/2/21_______________________________________________________________ Lab Time (circle): 8:00 am 9:50 am 12:40 pm 2:30 pm Data Complete the chart based on your observations. Isolation of casein 1. Mass of milk: ____20.85_____ g 2. Mass of filter paper: ___.455______ g 3. Mass of dried casein: ___2.716______ g Chemical analysis of proteins Buiret test Substance 2% glycine 2% gelatin 2% albumin Casein + H2O 1% Tyrosine Color Formed Clear (no purple) Darker Purple Light Purple Clear purple w/ no precipitate Clear (no purple) Positive or Negative? Negative Positive Positive Positive Negative Color Formed after Heating Very opaque, nearly black, purple Light clear “amethyst” Cloudy “violet” Clear “lavender” Dark & cloudy purple Positive or Negative? Negative Positive Positive Positive Negative Ninhydrin test Substance 2% glycine 2% gelatin 2% albumin Casein + H2O 1% Tyrosine Chemistry 1031 North Park University Discussion 1. Determine the percentage of casein in milk: % Case = mass dried C / Mass milk 200 ml x 100 = (2.716g/20.58) x 100 = 10.4% 2. Using the percentage you obtained for casein in milk, how many grams of casein are in a glass of milk (175 g)? Casein g = Milk g x % Casein = 175 g x .104 = 18.2 g Casein 3. Explain why casein precipitates when acetic acid is added to it. When acetic acid is added to milk, the pH changes from around 7 to about 4.6, which is also the isoelectric point of casein. This change in pH, which is swinging to the more acidic side of the pH spectrum, deprotonates the casein in the milk and causes it to form an insoluble precipitate which is the casein we collected in our experiment. There is no charge repulsion left in the solution, so the precipitate stays in its solid form, rather than emulsifying back into the milk. 4. Why is a protein more soluble when bearing an excess charge ( + or - )? As opposed to a nonpolar protein, a protein with excess charge can interact with other molecules in a solution thanks to charge repulson, which can either bind or break down a protein. 5. In order for the ninhydrin test to work, what functional group must be present? The ninhydrin test required a free NH2 group which would then interact with the ninhydrin.