Effect of pH on the rate of acid hydrolysis of keratin protein

Hair is probably one of the first things one notices about someone. Healthy and shiny hair always leaves an impression, and thus proper hair care is necessary. I recently started using a new shampoo under the influence of a super star who was advertising for it. A few weeks later, I realized that I was suffering from an increased hair loss than usual. My dermatologist suspected that this was because the pH of that particular shampoo was lower than usual. To further examine whether basic, acidic, or neutral nature is suitable for our hair, I decided to perform my Chemistry Internal Assessment on this topic. The pH of a solution used on hair has an effect on a protein called keratin present in hair. A holistic knowledge of Chemistry, specifically Acid and Bases, and Biology was necessary to explore this topic. The significance of my internal assessment is to find out ideal the pH of a shampoo to make sure keratin in hair is not subjected to any damage and thus hair remains healthy.

How does the average rate of acidic hydrolysis of the protein keratin depend on the pH at which it is carried out? Determined using a colorimeter.

Proteins: Proteins are condensed polymers. Chains of amino acids are linked to each other through peptide linkages to form proteins. Amino acids are called so as they contain both, the carboxylic acid group as well as the amine group. The formation of a protein can be explained thus: when 2 amino acids react with each other, the water molecule is eliminated and the 2 molecules condense to form a peptide linkage. In this way, the chain can be elongated to form more peptide linkages and that leads to the formation of protein. Thus, proteins are several amino acids linked to each other through peptide linkages.

Acidic Hydrolysis of proteins: Proteins undergo acidic hydrolysis where the peptide linkages are broken when the protein is treated with water in the presence of acid. Following this process, individual amino acids are obtained. In this particular experiment, the rate of acidic hydrolysis of the protein keratin as a function of pH, will be tested.

Biuret Test: Biuret test is the quantitative estimation of the components of polypeptides. The biuret reagent mainly consists of an alkaline solution of copper sulfate which is blue in color. The peptide linkages in protein chains react with the biuret reagent to form a violet complex which shows the maximum absorption at 550 nanometer.

Epsilon (ε), depends on the chemical used. Since the type of chemical is constant for all experiments, ε is a constant. The same apparatus (colorimeter) is used to measure the absorbance for all experiments, thus the optical path length (l) is constant. Therefore, absorbance directly relates to molar concentration such that, A ∝ c Thus, the molar concentration can be measured in terms of absorbance.

Focus of investigation - The investigation mainly focuses to determine the rate of acidic hydrolysis of keratin as a function of pH. This will enable us to understand what should be the ideal pH of the shampoo. If we can find the pH at which the rate of reaction is minimum, keratin will not be lost due to the acidic hydrolysis.

Experimental methodology - First, a human hair sample will be taken. Keratin will be extracted from the sample using ethanol and sodium carbonate. Following this, buffer tablets of different pH will be added to the solution containing extracted protein. A part of the solution will be pipetted out, to which the biuret reagent will be added. The absorbance of the violet complex obtained after adding the biuret reagent is calculated. The absorbance will then be measured at various intervals of time and a graph of absorbance against time will be plotted. The rate of reaction will thus be determined from the gradient of the curve.

Graphical determination of rate - The rate of reaction can be determined by \(\frac{∆c}{∆t}\) , where ∆c is change of concentration and ∆t is change in time. If concentration is plotted against time on a graph, then the gradient of the curve will give us the rate of the reaction.

ΔA - change in absorbance

ΔT - change in time

According to beer lambert law, absorbance is directly proportional to molar concentration, thus we can plot absorbance instead of concentration and hence the rate can be determined from the gradient of the absorbance v/s time curve.

This section refers to the research paper titled “Influence of hydrolysis temperature and pH on the selective hydrolysis of whey proteins by trypsin and potential recovery of native alpha-lactalbumin” published in the International Dairy Journal. The research is based on the effect of temperature and pressure on the hydrolysis of whey protein using bovine trypsin as an enzyme. Rate of the reaction was measure by the high performance liquid chromatography method. It was observed that above pH 8.5, the rate has started to decrease, while below pH 6.0 the rate was much higher.

The rate of hydrolysis of keratin protein has no correlaion with the pH at which it is being carried out. 

The rate of acid hydrolysis of the keratin protein has a correlation with the pH at which it is carried out.

• No forbidden chemicals were used.
• The methodology was designed in such a way that the experiments could be conducted with the minimum use of chemicals.
• Chemicals were diluted with tap water before disposing them in safe waste bins.
• No harmful gases where evolved in my methodology.
• The lab was kept clean by immediately wiping off any spilled chemicals.

• 2.00 ± 0.01gm of human hair sample was weighed on a digital mass balance using a spatula and a watch glass.
• The hair sample was dissolved in 50 cm³ of ethanol
• 5.00 ± 0.01gm of sodium carbonate was weighed on a watch glass using a digital mass balance and was added to the beaker, after which it was kept on a magnetic stirrer and operated for 30 minutes
• The solution was filtered and the filtrate was kept in a 100 cm³ beaker.

• A clean and dry 100 cm3 beaker was taken
• 50 cm³ of the protein extract was added to the beaker using a graduated measuring cylinder
• A buffer tablet of pH 2.00 was crushed into fine powder using a mortar and pestle and added to the beaker with the help of a spatula
• 2.00 ± 0.05 cm³ of the above solution was pipetted out using a graduated pipette and taken into a test tube
• 1.00 cm³ of copper sulfate solution was added to it
• 1.00 cm³ of NaOH was added to it
• A violet colored solution was obtained
• This solution was transferred into a cuvette and the absorbance of the solution was recorded at 550 nanometer using a colorimeter
• The same process was repeated for consecutively 5 more days