Venkatesh.T.et,al. International Journal of Technology and Engineering Science[IJTES]TM Volume 3[8], pp: 4008-4011, August 2015
Lab-Scale Extraction, Confirmation and Applications of Protein from Human Hairs Venkatesh.T1, Anbarasu.A2, Subramanian.O.S3 123
Department of Chemical Engineering, Coimbatore Institute of Technology, Coimbatore, Tamilnadu, India – 641014. 1
[email protected]
Abstract— This research work focuses on a simplified method of extraction of protein from human hairs, which inturn has many industrial applications. The extraction method includes the four steps: The hair samples are cut into smaller pieces, external lipids are removed using a mixture of methanol and chloroform, the hair samples are incubated in a buffer solution and finally the incubated materials are filtered through a gravimetric filter paper, from which proteins are removed from the filtrate. Buffer solution essentially consists of a mixture of Tris-HCl, Thiourea, Urea and mercaptoethanol. Experiment is repeated with various compositions of buffer solution, incubation time and incubation temperature. The results are optimized. The method is reproducible and reliable. Index Terms— Human hairs, Keratin-fibrous protein,
Extraction, Mercapto ethanol.
1. INTRODUCTION Proteins are biological molecules which are polymers of amino-acids[1]. Keratin is a form of protein which are mainly found in the human hairs. Keratin fills up around 70-85% of human hairs and one molecule of keratin measures 10-15 nm[2]. The primary component of keratin is sulfur because of the presence of the amino acid cysteine. It accounts for 24% of total amino acids present in the human hairs[2,3]. An enormous quantity of keratin is available in the form of hairs, horns, feathers etc.,[3]. As keratin is non biodegradable, it becomes necessary to utilize this protein in some forms and keratin has wide applications industrially and domestically. This paper deals with the simplified method of extraction of keratin, lab tests for confirmation and its applications. 2. EXPERIMENTAL PROCEDURE A. MATERIALS The materials required for performing this extraction is Urea, Thiourea, Tris-HCl and mercaptoethanol. All the chemicals are purchased from Mahalakshmi
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Chemicals, Coimbatore. Hair samples are collected from one volunteer so that we can minimize the experimental errors which may arise due to gender, age or other factors. The basic laboratory equipment and air dryer for heating has been provided by Coimbatore Institute of Technology, Coimbatore. B. METHODS It consists of 3 steps which are described below: I. PRETREATMENT OF HUMAR HAIRS Removal of external lipids is the first step towards the extraction of protein[4]. This can be done by immersing the hair samples in distilled water for one hour to remove any sort of impurities and then treating it with the mixture of chloroform and methanol for a prolonged period of 24 hours. During this period, chloroform/methanol mixture (50:50 W/W ratio) effectively removes the lipids in the form of liquid which can be seen as the oily layer on the top of the solution[4]. II. PREPARATION OF BUFFER SOLUTION: Buffer solution is a mixture of Tris-HCl, Thiourea, Based on urea and mercaptoethanol[4]. [5] stoichiometry , 0.25 M of Tris-HCl is prepared by adding 6.057 grams of Tris-HCl in water and making it to 200 ml. Similarly, 200ml solutions of Thiourea and urea is prepared by adding 39.58 and 60 grams respectively, thus making it to 2.6 M and 5 M respectively. 5% mercaptoethanol solution is made by adding 10ml of mercaptoethanol to 190 ml of distilled water. This 800 ml mixture of buffer solution is used for the extraction of protein. III. EXTRACTION Hair samples are now cut into smaller pieces of length 1-2 mm using knife or blades and this purified samples are now immersed in the buffer solution and kept inside the air-oven at the temperature of 50°C for a period of 24 hr. After 24 hours, the sample is taken out and filtered through a gravimetric filter paper and excluding the filtrate, the residue is the
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Venkatesh.T.et,al. International Journal of Technology and Engineering Science[IJTES]TM Volume 3[8], pp: 4008-4011, August 2015 obtained protein which is subjected to confirmatory tests.
10% mercuric sulphate in H2SO4 is added to the test solution and boiled. Yellow colour is obtained due to the precipitation of protein. Mercury combines with the tyrosine of protein which is identified by the formation of yellow colour. IV: NINHYDRIN TEST: Few drops of freshly prepared ninhydrin is added to the test solution and gently heated. Presence of glycine, an amino acid is confirmed by the appearance of violet colour. IV. SULPHUR TEST: This is the confirmation of Keratin. As keratin is mainly composed of cysteine, a sulphurous amino acid, this test is performed to confirm the presence of sulphur. Few drops of NaOH solution is added to the test solution, followed by the addition of lead acetate solution. Sulphur present in the keratin forms Na2S, which inturn reacted with lead acetate to form lead sulphide. This is confirmed by the appearance of black colour. All the above tests confirmed the presence of Keratin in the extracted product.
FIG I: EXTRACTED KERATIN
4. DISCUSSIONS
3. RESULTS
I. EFFECT OF CONCENTRATION
The extracted protein is subjected to the following confirmatory tests[6,7].
Tris-HCl is maintained at the same concentration of 0.25M as its role is to maintain the pH of the solution. Incubation time and incubation temperature is fixed at 24 hours and 50°C respectively. The concentration of other components of buffer solution is varied and maximum extraction is obtained with higher concentrations of mercaptoethanol.
I. BIURET TEST: Few drops of NaOH solution is added to 2 ml of test solution followed by the addition of few drops of CuSO4 solution. The test solution turns into violet or pink colour which is due to the presence of dipeptide linkages. Dipeptide linkages are the bonds which binds the protein molecules[8], and hence the presence of protein molecules is confirmed.
Thio-Urea
Urea
Mercapto ethanol
Yield
2.6M
2.5M
5%
58.9%
II. XANTHOPROTEIC TEST:
5.2M
2.5M
10%
61%
Few drops of concentrated nitric acid is added to the test solution and contents are boiled. Appearance of yellow colour during boiling indicated the presence of nitro derivatives of aromatic amino acids. The contents are then cooled and few drops of NaOH is added. The solution is turned orange which indicated the formation of sodium salts of nitro derivatives. This confirmed the presence of amino acid tryptophan, an essential amino acid of keratin molecule.
5.2M
5M
15%
64%
5.2M
5M
20%
64%
TABLE 1: EFFECT OF CONCENTRATION Yield is calculated by, Yield = (Amount of Keratin extracted in grams/Amount of hair samples incubated in grams) * 100
III. MILLONS TEST:
From this, it is clear that increasing the concentration of mercaptoethanol, increases the yield of keratin. It
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Venkatesh.T.et,al. International Journal of Technology and Engineering Science[IJTES]TM Volume 3[8], pp: 4008-4011, August 2015 is attributed to the fact that disulphide bonds of proteins are cleaved to the greater extend in presence of mercapto ethanol which facilitated the separation of keratin from human hairs. Decreasing the content of urea and thio-urea reduced the extraction of keratin and thus it is concluded that increasing the content of mercaptoethanol increases the yield of protein with adequate concentrations of thio-urea and urea. II. EFFECT OF TIME The buffer solution of concentration corresponding to maximum yield is fixed and incubation time is varied. It is found that yield increased with increase in time upto 25 hours, after which it remained constant. Hence optimum time of 24 hours (1 day) has been chosen.
It is attributed to the fact that rate of a reaction increases with increase in temperature[8], optimum temperature of 50°C has been chosen. 5. APPLICATIONS Keratin has many industrial and domestic applications. It can be used as a protein feed stock for cattles[9]. It is also reported that keratin can be used to manufacture organic fertilizers, which enhances the plant metabolism[10] . It can also be used for preparing cosmetics which improves hair growth and skin conditions[11]. Keratins have the property of selfassembly and thus it can be extensively used in the field of drug delivery systems, tissue engineering and wound healing[12]. Keratin hydrolysates has potential applications in leather tanning industries. Keratin can be converted into Keratin hydrolysates through alkali hydrolysis which inturn can be used in leather tanning process[3]. This usually eliminates the use of chromium and chromium sulphate in leather industries, which is toxic in nature. 6. CONCLUSIONS
FIGURE 2: EFFECT OF TIME Maximum yield of 64% is obtained at 25 hours and hence optimum time of 24 hours is chosen. III. EFFECT OF TEMPERATURE Temperature is varied accordingly by keeping the other parameters constant and it is found that yield increases with increase in temperature upto 50°C.
Thus, maximum yield of 64% of keratin is obtained at the imcubation temperature of 50°C, time period of 24 hours and a buffer solution containing Tris-HCl, Thiourea, urea and mercaptoethanol. Despite having enormous applications, larger amount of keratin is wasted globally. It is available in larger numbers in the form of human hairs, poultry wastes etc., Research is being carried out globally to utilize this keratin into useful products. REFERENCES [1] Advanced organic chemistry, Arun Bahl and B.S.Bahl, ISBN 13: 9788121935159. [2] Structure and chemistry of Keratin fibers, Bradbury JH, Adv Protein Chem, 27(1973) 111211. [3] Industrial applications of keratin – A review, R.Karthikeyan et al., Jr of Scientific and Industrial Research, Vol 66, September 2007, 710-715. [4] A Rapid Extraction Procedure of Human Hair Proteins and Identification of Phosphorylated Species, Akira Nakamura et al., Biol. Pharma. Bull. 25(5) 569-572 (2002). [5] Chemical Process Calculations, D.C.Sikdar, ISBN-13:978-8120347823. [6] Biochemical Methods by S.Sadasivam and A.Manickam,Second Edition,New Age International Publishers,Page-22.
FIGURE 3: EFFECT OF TEMPERATURE
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Venkatesh.T.et,al. International Journal of Technology and Engineering Science[IJTES]TM Volume 3[8], pp: 4008-4011, August 2015 [7] Lab Manual in Biochemistry, Immunology and Biotechnology, Arti Nigam and Archanaayyagari, Tata McGraw-Hill Education. [8] Chemical Reaction Engineering, Octave Levenspiel, ISSN: 978-0471254249. [9] A biotechnological process for treatment and recycling poultry feathers as a feed ingredient, A.Bertsch et al., Bioresource Technology, 96 (2005) 1703-1708. [10] Biodegradation of keratin waste: Theory and practical aspects, Kowalska Teresa et al., Waste Management, 31 (2001) 1689-1701. [11] Brazilian keratin hair treatment: a review, Journal of Cosmetic Dermatology, Vol 12, Issue 2,Pages 144-148, June 2013. [12] A Review of Keratin-Based Biomaterials for Biomedical Applications, Jillian G. Rouse et al., Materials 2010, 3, 999-1014.
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AUTHOR BIOGRAPHY 1.
2.
3.
VENKATESH T, completed his B.Tech in Chemical Engineering from Coimbatore Institute of Technology, Coimbatore. He is currently pursuing his M.Tech Chemical Engineering from Indian Institute of Technology, Guwahati. He is about to complete his M.Tech degree in the academic year 2015-16. ANBARASU A, completed his B.Tech in Chemical Engineering from Coimbatore Institute of Technology, Coimbatore. He is currently working as an executive officer in Asian Paints, Cuddalore. He is also preparing for civil service examinations and he is an IAS aspirant. SUBRAMANIAN OS, completed his B.Tech in Chemical Engineering from Coimbatore Institute of Technology, Coimbatore. He is currently working as a process engineer in Manali Petrochemicals, Chennai.
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