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

• On dissolving the human hair samples in the ethanol layer in the presence of sodium carbonate, bubbles were observed.
• The above mentioned solution became cloudy.
• It was filtered to obtain a clear solution.
• The intensity of the violet color in the solution at every pH was decreasing as time increased, which means that the polypeptide in the solution was decreasing.

Sample calculation:

For Day-0,

Average =\(\frac{0.329+0.328+0.329+0.324+0.329}{5}\) = 0.328

Standard deviation

= \(\frac{(0.329−0.328)^2+(0.329−0.328)^2+(0.329−0.328)^2+(0.324−0.328)+(0.329−0.328)^2}{5}\) = 0.002

The independent variable, pH of solution, has been plotted in the x-axis. Rate has been plotted in the y-axis as it is the dependent variable. Concentration is measured in terms of absorbance and time is measured in terms of days, thus the rate has been measured in terms of absorbance per day.

We can see that rate decreases from 11.50 abs day^-1 × 10^-2 to 7.60 abs day^-1 × 10^-2 in acidic region as the pH increases. In the basic region, rate decreases from 5.90 abs day^-1 × 10^-2 to 3.10 abs day^-1 × 10^-2 as the pH increases.

As the pH increases, the acidity of the medium decreases and so does the rate of reaction. Thus it can be concluded that the reaction is favored in acidic medium. As the pH is increasing, the medium becomes more alkaline, the rate decreases. Thus this data also confirms that reaction is favored in an acidic medium.

Peptide linkages or amide bonds can be broken by amide hydrolysis, where the linkages are cleaved due to the addition of water molecule. The metastable peptide bonds will break spontaneously in a slow process. The H⁺ ion in the water molecule will facilitate the reaction between carbon and oxygen molecule, thus forming a tetrahedral intermediate. The intermediate ultimately dissociates to carboxylic acid and ammonium salt.

The H⁺ ion facilitates the breaking of the carbon nitrogen bond. As pH is increasing, the concentration of H⁺ ions increases, and rate increases because hydrolysis of linkage is facilitated.

Since there was no observed trend, no trend line has been plotted in the graphs. The value of regression coefficient cannot be calculated as the data does not belong to any specific category or is not continuous (values in the x-axis are not continuous). Thus, we cannot do any statistical analysis in this graph.

The basic aim of my investigation was to understand how the rate of acidic hydrolysis of the protein keratin depends on the pH at which it is carried out. It has been very clearly observed that the rate of the reaction of acidic hydrolysis of keratin protein at pH 2.00, 4.00, 9.00 and 11.00 are 11.50 X 10^-2 , 7.60 X 10^-2 , 5.90 X 10^-2 , and 3.10 X 10^-2 in terms of absorbance per day respectively.

Although there is no evident trend in the data, it is very clear that rate differs with pH. As the pH increases from 2.00 to 4.00, the rate changes from 11.50 X 10-2 to 7.60 X 10-2 . Thus, in the acidic region, as the pH becomes less acidic the rate decreases. As the pH increases from 9.00 to 11.00, the rate changes from 5.90 X 10-2 to 3.10 X 10-2 . Therefore, in the basic region, as the pH increases and the medium becomes more basic the rate of reaction decreases. From this we can conclude that the reaction is favored in the presence of H⁺ ions, or an acidic environment. As the medium becomes more acidic, the hydrolysis occurs at a faster rate. It is also seen that as the pH increases, the medium becomes less acidic and the rate decreases.

The qualitative observations are in agreement with the above made conclusions. As we move towards a higher pH value, the intensity of the violet color obtained was lesser, and the time taken for the violet color to change its intensity was also more. The lower intensity of the violet color indicates that polypeptide in the solution is decreasing, which supports the conclusion.

Since there are significant differences in the values for the rate against the pH, the null hypothesis is rejected and the alternate hypothesis is accepted.

The scientific explanation to this is the hydrolysis mechanism of amides. Breaking of the peptide bonds involves the H⁺ ions in the mechanism. Oxygen donates its lone pair to H plus ions and gets protonated, leading to the formation of an intermediate. Thus the carbon nitrogen bond is broken. If medium is more acidic, the mechanism is favored. Intermediate is formed more readily and the hydrolysis occurs at a faster rate

• The investigation is done to mimic realistic situations:
  • Buffer tablets were used to vary the pH instead of using any other reagent because shampoos are manufactured using buffer tablets.
  • Since the degradation process is not very fast, instead of measuring the rate in terms of minutes, seconds or hours, it is measured in terms of days.
• Multiple trials were performed to minimize random error. The standard deviation was calculated which was very low in comparison to the magnitude of the data collected. Data collected has decimal places while standard deviation has a positive integer only in the 3^rd decimal place. Low values thus indicate that the data collected has high precision.
• A wide range of values were taken. Data was collected over a period of days and therefore there are data points in each of the raw data curve using which the rate of the reaction is calculated. This makes the measurement of rate more accurate. The values of regression coefficient are quite high in the raw data curve where absorbance is plotted against time. This means that the linear plot which has been done is quite accurate

Random error: The uncertainty of apparatus used and human error. It was minimized by taking multiple readings and taking the average.

Systematic error: The colorimeter will have an instrumental error associated with it due to fluctuation of voltages at which it is operating. This was minimized by calibrated the colorimeter before using it. Distilled water was taken in the cuvette and put it in the colorimeter. The wavelength was fixed at approximately 530 nanometer and the absorbance was made

• Sodium carbonate is used to extract keratin from the hair sample. It is alkaline in nature and thus can have a harmful effect on the keratin and cause it to degrade even before the reaction has started.
• Protein has been extracted in an aqueous medium where the protein is not highly soluble in water layer.
• Instead of using a biuret reagent in the experiment, we can also study the degradation of keratin using high performance liquid chromatography or spectroscopy. Due to the unavailability of these instruments in the school laboratory, this methodology was not carried out.

 Sampling error: Hair might differ in composition as well as the amount of keratin content in them. To avoid the sampling error, it was made sure that all the samples were collected from the same source, that is from the same person, and the same area of the skull in order keep them biologically same

Further scope of investigation: I would also like to study the effect of pH on the rate of denaturation of the protein albumin. The methodology that could be followed is stated below:

• Fluid inside the egg is taken in a beaker.
• Water is then added to it, along with different buffer tablets of different pH.
• The samples are heated at the same temperature.
• The time required for the mixture to solidify is then noted down
• The time taken can be used to measure the rate of the reaction and thus conclusions about the effect of pH on denaturation can be made.

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