1 THE ANALYSIS OF PROTEASE ENZYME ACTIVITY BY THE FORMAL TITRATION OF AMINO ACIDS BY: NI PUTU MEYAN PRATIWI (1013031025) University Student of Chemistry Education, Faculty of MIPA, Undiksha Abstract Amino acids are compounds that have one or more carboxyl group (-COOH) and one or more amino group (-NH2). Amino acids obtained from hydrolysis of proteins. Protein hydrolysis can be performed using the protease enzyme of trypsin. In this experiment aims to hydrolyze proteins in gelatin into smaller parts with the enzyme trypsin and make the relationship between the volume of NaOH and time and the curve mg amino acid nitrogen against time. The method used is the formal titration of amino acids. The results of this experiment showed that the enzyme trypsin can hydrolyze gelatin into parts smaller. For the relationship between the volume of NaOH toward time and the curve mg amino acid nitrogen against time showed the same trend, i.e. the longer the incubation time more volume of NaOH used to titrate and the more nitrogen mg obtained. Key words: amino acids, gelatin, enzymes, hydrolysis, titration of amino acids formal. To overcome this problem, formaldehyde should be added to a solution of amino acids to react with amino groups are uncharged, allowing the buffer process of ammonium groups in the region of lower pH and can be titrated at the end point quantitatively by using an indicator (Tika, 2010). The formal titration, as described by Seronsen, has been most useful in the determination of amino-acids and especially (HOCH2)2NCHRCOO in following the course of hydrolysis of H2NCHRCOO + 2HCHO proteins. The method as originally described In this experiment created amino acid could not be used for accurate determinations titration curve obtained from the hydrolysis of small quantifies of amino-acids owing to of protein by using protease enzyme. During the difficulty of determining the exact endthe hydrolysis of a protein, a number of point. The value obtained also depends on carboxyl and amino groups are increase. the point at which the titration is started Quantitative determination of one group will (Northrop, 2011). be able to give an indication to determine the In addition, Seronsen observed that degree of hydrolysis of a protein (Tika, when the amino acid solution was added a 2010). solution of formaldehyde, amino acid According to the zwitterions theory, if solution will be more acidic than the one amino acid in the solution is titrated with original. The addition of formaldehyde a base, means that the hydrogen ion from the produce hydroxymethyl derivatives. These ammonium group is titrated. Ammonium derivatives have the properties that form a group of amino acids are buffered at high stronger acid than the original compound pH, above pH 11, so it may be titrated to an (Poedjiadi, 2005). end. The same is true of the carboxyl group Reactions that occur in the titration is a buffer at low pH so it may also be using formaldehyde are reaction of forming titrated with base (Tika, 2010). dimetilol. Protein solution was neutralized Introduction A protein if the hydrolyzed would be obtained amino acids constituent. Protein hydrolysis can be performed using protease enzymes than trypsin will produce free amino acids. This reaction causes the isoelectric form of free amino acids lose a proton from the group NH3+ (Tika, 2010). Reaction: H3N + CHRCOO H2NCHRCOO - + H+
2 with a base (NaOH), was added to form dimetilol. With the formation of this dimetilol, means the amino group is bound and will not affect the reaction that occurs between the acid groups (carboxyl) with base (NaOH) so that the end point of titration can be properly terminated. Indicator used is phenolphthalein. If the right end point of the titration is obtained, then the solution will change color from clear to pink that does not disappear within 30 minutes. Enzymes are catalytic proteins. As a protein, enzyme has a specific condition in which the enzyme can work optimally, because the environment supports the most active conformation of the enzyme molecule. Temperature is one important environmental factor in the activity of an enzyme. (Campbell, 2000). Materials and Methods
100 mL erlenmeyer (this solution is used as a control or the "0" min). Boiled to destroy enzymes and then chill. Add 15 ml of neutral formalin and 3 drops of phenolphthalein. At time intervals 15, 30, 45, 60, 75 and 90 minutes from time to "0" do the same thing as was done in control. At each of the above reactions do titration with 0.02 M NaOH solution until the pink color is end point. Results and Discussion In this experiment performed a titration of amino acids, which egg solution is used as a sample of trypsin and protease enzymes to hydrolyze these proteins into smaller parts. Egg solution is then added with 5 drops of PP and 8 drops of 0.2 M NaOH, the addition of NaOH is stopped when a solution of was pink.
The materials that use in this experiment are 20 mL of egg solution, 15 drops NaOH 0.2 M solution, 150 mL of NaOH 0.02 M solution, 20 drops of HCl 0.1 M solution, 27 drops of PP 1% indicator, 15 mL of Formaldehida 40% solution, 25 mL of trypsin 1% solution and 100 mL of Aquades. Methods: 100 mL of egg solution was added 1 mL of phenolphthalein and 0.2 M NaOH solution dropwise until the pink color arises. After 0.1 M HCl was added drop by drop until the pink color just was missing (pH 8). Furthermore, the solution has been given treatment gelatin is soaked in water at a temperature of 38°C incubator for 5 minutes. On the other tube, 25 mL of trypsin solution was added a few drops of phenolphthalein and drop by drop a solution of 0.2 M NaOH until the pink color arises. 0.1 M HCl was then added dropwise until the pink color just was missing (pH 8). Subsequently incubated in an incubator at a temperature of 38°C water for 5 minutes. The second solution was incubated for 5 minutes was then mixed and stirred slowly. When the mixing time is calculated by using a stopwatch. Once mixed evenly taken 10 mL of the mixture, put in a
Fig 1. Egg solution
Fig 2. Egg solution that added PP and NaOH
Furthermore neutralized with 0.1 M HCl until the pink color disappears, with added 12 drops of 0.1M HCl solution. The goal of treatment is to condition that the solution reached pH 8, and immersed in an incubator at a temperature of 38°C. The same was done on the trypsin solution. Taken 5 mL of trypsin solution and was added 5 drops of as much PP and 2 drops of 0.2 M NaOH until solution become pink. Furthermore the acid is neutralized with 3 drops of 0.1 M HCl to form a solution with pH 8 and immersed for 5 minutes in the incubator with the temperature maintained 38°C.
3
Fig 3. Mixture of tripsin, PP, and NaOH.
Fig 4. After adding HCL become yellow
The purpose of the conditioning egg solution and trypsin at pH 8 is that when the two solutions are mixed to keep pH 8 and pH 8 is the optimum temperature for enzyme activity of trypsin. Likewise with incubation at 38°C intended to allow the enzyme trypsin may be at an optimum temperature so that the activity of enzyme will maximum. When the incubation time of egg solution and trypsin solution was 5 minutes, then the two solutions are mixed and the timing started. When the start of mixing was calculated as the minute is 0 and 2 mL of the mixture was immediately taken to do the remaining solution back titration and incubated at 38°C temperature. Before the solution was titrated first enzyme destruction done by heating the mixture above the heater. Then cooled, after a new cold solution was added with 3 drops of PP and 5 mL of formaline. Subsequently titrated with NaOH 0.02 M. In the titration of amino acids to determine the ability of the enzyme trypsin degrade or hydrolyze gelatin into parts smaller again. The principle of this experiment, the protein follows the zwitter ion theory is an amino acid has two active sides (-COOH group and the amine groupNH2), so it can be titrated using acids and bases. But in reality, the ammonium group of the amino acid is a buffer at high pH (above pH 11) so it is not possible to titration at the end point of the indicator. This is because an indicator generally has a pH scale as far as possible close to pH 7 due to acid-base titration is a neutralization reaction. Similarly, the carboxyl groups in amino
acids, a buffer at low pH so it is not possible also to titration. To anticipate this, then this is where the role of the addition of 40% formaldehyde. The addition of formaldehyde 40% in the titration function for carbonyl group reacts with amino acids that are not charged to allow the group to react with NaOH and ammonium buffer at a lower pH. When a solution of egg and trypsin from mixed, then that's when egg solution is hydrolyzed by the enzyme from trypsin. Where the enzyme trypsin to cut at the end C of arginine, lysine or amino ethyl cysteine. If Rn+1 contain a proline then the cutting edge C can not be performed (R = arginine, lysine, amino ethyl cysteine). -Ala-Gly-Pro-Arg-Gly-Glu-4Hyp-Gly-ProSubsequently carried out every 15 minutes is taking a mixture of egg solution and trypsin. Treated the same as the performing of titrations at minute 0. Starting from the destruction of the enzyme, cooling, addition the PP indicators and formalin and than titrated with NaOH 0.02 M. Titration was stopped when the solution has turned into a little red.
Fig 5. After titration color of solution become pink
4 Below is data that obtained after titration. Table 1. Data of relationship between time and volume of NaOH
0
Volume of NaOH (mL) 5.5
15
5.9
30
6.4
45
7
60
7.7
75
8.5
90
9.4
Time (minutes)
Based on data above can be made curve titration between time and volume of base. Titration curve between time and volume of NaOH (mL)
volume of NaOH
25 20 y = 0.0433x + 5.25 R² = 0.9826 15 10 5 0 50 100
volume NaOH
Time (minutes)
Based on these graphs, it can be a tendency that the longer a mixture of egg solution and trypsin incubated, the more the volume of NaOH required to titrate the amino acid solution. This can occur because during the hydrolysis of egg solution by trypsin produced a number of amino acids that have an increasing concentration with increasing contact between the solution of egg solution and trypsin. Calculation V NaOH t (9.4 5.5) Degree of hydrolysis 0.043 (90 0) Degree of hydrolysis
Assuming 1 mL of NaOH is equivalent to 1.4 mg amino acid nitrogen, it can count the number of amino acids in each volume of NaOH as the titration with the following calculation. 1 mL NaOH 0.1 N equivalent to 1.4 mg amino acid nitrogen, so: 0.02 X 0.1 1.4 0,1X 0.028 X 0.28 mg Thus 1 ml of 0.02 N NaOH solution equivalents to 0.28 mg amino acid nitrogen, which can be calculated mg of nitrogen that is used for each time interval titration. Calculation: At 0 minute , VNaOH = 5.5 mL, so 5.5 x 0.28 = 1.54 mg At 15 minutes, VNaOH = 5.9 mL, so 5.9 x 0,28 = 1.65 mg At 30 minutes, VNaOH = 6.4 mL, so 6.4 x 0,28 = 1.79 mg At 45 minutes, VNaOH = 7 mL, so 7 x 0,28 = 1.96 mg At 60 minutes, VNaOH = 7.7 mL, so 7.7 x 0,28 = 2.15 mg At 75 minutes, VNaOH = 8.5 mL, so 8.5 x 0,28 = 2.38 mg At 90 minutes, VNaOH = 9.4 mL, so 9.4 x 0,28 = 2.63 mg Appropriate with calculations, data mg of amino acid for each volume of NaOH can be written in the following table. Table 2. Data of relationship between time and mg amino acid nitrogen Time (minutes) 0
Volume mg Amino acid of NaOH nitrogen (mL) 1.54 5.5
15
5.9
1.65
30
6.4
1.79
45
7
1.96
5 60
7.7
2.15
75
8.5
2.38
90
9.4
2.63
From the table, can be made curve relationships mg amino acid nitrogen to time as follows. Curve of mg Nitrogen Amino acid toward time mg of amino acid
3
y = 0.0121x + 1.4689 R² = 0.9821
2 1
massa mg
0 0
50
100
time (minutes)
Conclutions Based on the discussion above, can obtained several conclusions, among others: 1. Protein can be hydrolyzed by protease enzyme from trypsin. 2. From the data of result titration of amino acid can be made curve the relationship between volumes of NaOH with time. From curve can be known the amount of volume NaOH that use is directly proportional with interval of time. 3. From the relationship mg nitrogen with time curve can determine the value of mg nitrogen is directly proportional with time. Acknowledgment The author thank to I Nyoman Tika, M.Si and I Ketut Lasia for constant encouragement. The author thank to Made Eka Guna Tresna and I Wayan Gde Oka Prabawa, assistant of biochemistry in chemistry department, Ganesha University of education and the author thank for all of students in Pioneering chemistry department, Ganesha University of education.
References Campbell, Neil A, et al. 2000. Biologi. Edisi Kelima, Jilid 1. Judul Asli Biology, Fifth Edition. Penerjemah Dra. Rahayu Lestari, dkk. Jakarta: Erlangga. Northrop, John H. 2011. A Convenient Method For The Formal Titration. Accessed at 5th April 2013. Poedjadi, Anna dan Titin Supriyanti. 1994. Dasar-Dasar Biokimia. Jakarta: Universitas Indonesia Tika, I Nyoman. 2010. Buku Penuntun Praktikum Biokimia. Singaraja: Universitas Pendidikan Ganesha.
