THE EFFECT OF INHIBITOR TOWARD ENZYME ACTIVITY

Enzymes are proteins that catalyze chemical reactions in biological systems in terms of the biocatalyze which has the nature of catalysts in general are involved react, but at the end of the reaction will be retrieved in its original form. Inhibitor is a compound that can inhibit or decrease the rate of reaction catalyzed by the enzyme. Enzyme inhibitors itself are divided into two kinds namely reversible and irreversible inhibitor. The aims of this experiment was to determine the effect of inhibitors on enzyme activity seen from the value of V_max and K_M reaction without inhibitor and with inhibitor. The method used was quantitative analysis. In this experiment were used sodium succinate as a substrate and sodium citrate as a inhibitor. The Na-citrate was inhibited reaction catalyzed by the enzyme succinate dehydrogenase. The value of K_M  in reaction without inhibitor is 6.754 x 10^-2 M, while in reaction with inhibitor is 0.4 M. So, it can be conclude that addition of inhibitor concentration cause increasing in K_M value but had no effect on the value of V_max..

Keywords: Enzyme, Succinate dehydrogenase, Competitive inhibitor

THE KINETIC OF ENZYMATIC REACTION

Enzymes are proteins that catalyze chemical reactions in biological systems in terms of the biocatalyze which has the nature of catalysts in general are involved react, but at the end of the reaction will be retrieved in its original form. The aims of this experiment was to determine of the value of V_max and K_M using the relationship curve between the enzymatic reaction rate and substrate concentration.  The method used was quantitative analysis. Then, the instrument used was Spectronic 20⁺. In this experiment were used five test tubes for each 20 minutes and 0 minute of incubation. In this case, the substrate used was casein and the enzyme used was trypsin. The kinetics of enzyme reactions can be determined by calculating the value of K_M and V_max. The value of V_max and K_M are determined by using the relationship curve between the rates of enzymatic reaction with substrate concentration The result of this experiment is the value of K_M and V_max for the enzymatic reaction with the enzyme trypsin and casein substrate with t = 0 minute and t = 20 minutes were 0.424 mg/mL and 0.518 mmole/minutes. 
Keywords: Enzyme, Anson method, Lineweaver-Burk plot

DETERMINATION OF PROTEIN CONTENT IN EGG ALBUMIN SOLUTION THROUGH LOWRY METHOD

Egg albumin is one sources of protein. Determination of the concentration of protein is a process routinely used in chemical analysis. One purpose of determining the concentration of protein is to know the nutritional value of a food ingredient. There are several methods to determine the protein concentration, such as the Biuret method, Lowry method, and so forth. The objectives of this experiment was to determine the protein content in egg albumin solution through Lowry method.  The method used in this experiment was quantitative analysis. The reagent used was Biuret reagent and Folin-Ciocalteu reagent. Folin-Ciocalteu reagent can detect tyrosine residues because it can  reduce fosfotungstat and forfomolibdat becomes tungstate and molybdenum which has blue color. Biuret reagent also lead the reduction reaction. In determining the protein content of sample, it beginning from making the calibration curve between concentration and absorbance and it got the linier formula y= 0.0461 x + 0.0441 with R = 0.9614. Based on the experiment result, it can be concluded that the content of protein in sample of egg albumin solution is 0.352 mg/mL (in 100 times dilution). Therefore, the initial concentration of the sample is 35.2 mg/mL.

Keywords: Egg albumin, Lowy method, Follin-Ciocalteu reagent, Biuret reagent

THE TEST OF PROTEIN IN EGG ALBUMIN SOLUTION

Proteins are polymers of amino acids that linked through a peptide bond. Egg albumin is one of the main sources of protein. The objectives of this experiment were (1) to identify protein derived from egg by utilizing a unique bond in the protein which is peptide bond by several protein tests, and (2) to determine the effect of physical changes caused by the changing of  temperature, pH and chemical substances on the structure protein The method used in this experiment was qualitative method. Based on the result obatined, it can be concluded egg albumin solution contains protein and can be identified by several tests. The results were Biuret test showed positive results, namely a purple solution due to the formation of complex Cu²⁺; precipitation of proteins by metal HgCl₂ and Pb(CH₃COO)₂ produced white precipitate;  precipitation by salt (NH₄)₂SO₄ which produced white precipitate and its precipitate were positive in Millon test (red precipitate) and soluble in water, and then the filtrate was positive with the Biuret test (bluish solution); precipitation of proteins by alcohol produced white precipitate in the acid and buffer conditions; protein coagulation produced  white precipitate and positive in Millon test (red precipitate); and protein denaturation produced white precipitate in acid and buffer conditions. From the test that has been done,  egg contains the amino acid tyrosine, as shown on the Millon test that is specific to this amino acid. Then, the structure of protein was influenced by temperature, chemical substances and pH.

Keywords: Amino acids, Egg albumin, Biuret test, Protein precipitation by metal, Protein precipitation by salt, Coagulation test, Protein precipitation by alcohol, Protein denaturation 

IDENTIFICATION OF AMINO ACID IN UNKNOWN SAMPLE THROUGH PAPER CHROMATOGRAPHY TECHNIQUE

Chromatography is introduced as a technique for separating and/or identifying the components in a mixture. One type of chromatography is a paper chromatography, which has the stationary phase and mobile phase that the liquid which is not mixed with each other. By using paper chromatography, it can be determined amino acid content that is existed in the sample by comparing the value of Rf sample components with the value of standard Rf. On paper chromatography experiment was used a mixture of n-butanol, aquades, and glacial acetic acid as the eluent. In this experiment, it was carried out a qualitative analysis of a solution containing several amino acids, which aims to determine the ratio of the distribution coefficient (R_f) from a variety of amino acids, especially glycine, cysteine, phenylalanine and tryptophan as well as determination on unknown sample by paper chromatography in ascending technique. Based on the experiment, the R_f value of glycine, cysteine, trypthopan, phenylalanine, unknown A, and B solutions namely 0.186; 0.197; 0.569; 0.605; 0.174; 0.232; 0.174; 0.558 and 0.581. Then, the amino acids that contained in the unknown A were glycine and cysteine. While, the amino acids that contained in the unknown B were glycine, tryptophan and phenylalanine.

Keywords: Amino acid, Paper chromatography

HYDROLYSIS OF GELATIN BY PROTEASE ENZYME THROUGH FORMAL TITRATION METHOD

Proteins are organic compounds consisting of one or more amino acids. If the protein is hydrolyzed, it will be produced amino acid. One way that can be used to obtain the amino acid is hydrolysis of protein. The aims of the experiment were (1) to hydrolyze protein with protease enzyme, and (2) to make curve between NaOH(mL) vs time (minute) as well as curve of nitrogen amino acid mass (mg) vs time (minute) by formal titration method. This experiment used quantitative method. In this experiment, amino acids obtained from hydrolysis of gelatin solution by the enzyme trypsin (protease enzyme) was titrated by NaOH solution. It should be noted also the condition of trypsin and gelatin solution were maintained by incubated at 38° C and pH=8 for reactions run optimally. The result of this experiment was protein hydrolysis of gelatin can be done by using protease enzyme. Then, curve between volume of NaOH that was needed in titration and time that was used by gelatin solution to react with protease enzyme was directly proportional. After that, curve between the mass (mg) nitrogen of amino acid that was produced and time that was used by gelatin solution to react with protease enzyme was directly proportional.

Keywords: Amino acid, Formal titration, Protease enzyme

IDENTIFICATION OF AMINO ACID IN EGG ALBUMIN, UNKNOWN B AND D SOLUTIONS THROUGH MILLON, HOPKINS-COLE, NINHIDRIN, PbS, AND NITROPRUSIDE TESTS

Amino acid is a compound that is very important as a constituent unit of protein. Total amino acids exist in nature, only 20 types of amino acids contained in protein. The purposes of this experiment are to identify (1) the types of amino acids contained in protein solution (egg albumin) through Millon, Hopkins-Cole, Ninhydrin, PbS and Nitropruside test, and (2) the types of amino acids contained in the unknown sample through the amino acid test. The method used in this experiment was qualitative analysis. The result of this experiment were, first, egg albumin solution is positive in Millon, Hopkins-Cole, Ninhydrin, and PbS tests. It means that in egg albumin contains tyrosin, tryptophan, α-amino acid and sulfur. Second, unknown B solution is positive in Millon test, in which it contains hydroxyphenyl compound. Besides that, unknown B solution is also positive in Ninhydrin test in which it contains α-amino acids and peptides having free α-amino group. While, unknown D solution is positive in Nitropruside test, thus it contained –SH group. So, it can be concluded that egg albumin contains tyrosin, tryptophan, α-amino acid and sulfur; unknown B is tyrosine solution; and unknown D is cysteine solution.

Keywords: Amino acids, Egg albumin, Millon test, Hopkins-Cole test, Ninhydrine test, PbS test, Nitropruside test.