Etiket Arşivleri: Laboratory‎

Laboratory‎ > ‎Dialysis v1

Purpose: In this experiment, the dialysis experiment is made. Dialysis is a process that is based on the principle of osmosis, moving from an area of high concentration to a lower concentration. Therefore this laboratory is for observing the enzyme purification.

Materials: Dialysis bag, beaker, magnetic stirrer, stirring bar, water, lugol solution, benedict solution, ninhydrin solution, biuret solution, pipette, rope

Procedure: Firstly, one dialysis bag was filled with a mixture of consisting of quantities of a 2ml of %40 glucose and 2ml of %5 starch, and other dialysis bag, 2ml %5 BSA and 2ml 10% Glycine. After making this action, the content of the bag cannot escape by leaking, knotting in end of dialysis tubing. Then bags were putted into beakers contains distilled water and waited about 2 hours.

Laboratory‎ > ‎Gel Filtration Using Sephadex

In this experiment we applied gel filtration of enzyme b using sephadex as column material. This is a chromatographic technique used for protein purification. The principle is based on fractionation of proteins based on the molecular weight or size of protein molecules. In this system when the mixture of the protein is applied at the top of the gel filtration column and washed with buffer solution, depending on molecular exclusion limit of column material, the large protein molecules are excluded from the internal volume and thus leave the column first. The smaller protein molecules are absorbed by the pores of the column material and leave the column later.

We collected 24 fractions from the column but 16 of them are illustrated at the data. Because the other fractions couldn’t measured correctly. From these data we calculated the protein content, specific activity, purification fold and total activity of each fraction. The results show that 4th and 6th fractions have the higher activity. Also the protein content and total activity versus fraction number graphic is plotted. At this graphs the first peak illustrates the first eluted fraction and this is probably eluted from the void volume also from the second graph we can see that the firstly eluted fractions activity are lower.

QUESTIONS

1. Which fraction is the most active?

– 4th and 6th fractions are most active since their total activity is equal to each other and higher than the other fractions.

2. Calculate specific activity, purification factor, % recovery.

– Calculations are done at the calculation section.

3. Which fraction has the highest purity?

– 11th fraction has the highest purity since it has the highest purification fold.

Laboratory‎ > ‎Heat Treatment

In this experiment we investigated the effect of temperature on enzyme activity and protein content. After the calculations we plotted two graphs these were protein content versus temperature and total activity versus temperature. According to the graphs we can say that the total activity increases up to a point then it starts to fall down. Initially this increase is caused by the increase in temperature up to the optimum temperature for reaction to gain sufficient energy to reach the activation energy. Then the activity decreases since further increase in temperature cause denaturation of protein. According to the graph the optimum temperature is about 400C. Also at that temperature the purity of the enzyme is highest. In contrast to total activity, protein content decreases down to a point then it starts to increase.

QUESTIONS

1. State the temperature at which the activity is highest.

– Activity is highest at about 400C

2. State the temperature at which the purity is highest.

– The highest the specific activity, the highest the purity. So at 400C purity is highest.

3. What is the use of purification? Does it always necessary?

– Purification involves removing any contaminants that are present in the mixture; these may be other enzymes or proteins, carbohydrates and ash. For the some determinations it is necessary to purify enzyme from other substances in the case of determination amino acid composition, molecular weight, tertiary and quaternary structure, substrate specificity and kinetic

parameters like Km, Vmax, kcat.

4. Under which conditions a purified enzyme is required for activity determination?

Laboratory‎ > ‎Immobilization v1

PURPOSE:

The aim of this experiment is compering the reaction rate of immobilized enzyme and free enzyme to find the effectivness factor

PROCEDURE:

First of all 1 ml olive oil and 3 ml 25 mM KP buffer ( pH = 7.0 ) were putted into the flask and then 12 mg immobilized enzyme (lipase) was putted and mixed. After that ıt was placed into water bath at 37 ºC for 30 min. After 30 min the flask was centrifuged for 1 min and supernatant taken and ethanol was added on it. However, the time passed from starting centrifuge to end of adding ethanol was recorded. And then phenolphthalein was added as indicator and titrated with KOH( 0,05 N).

Laboratory‎ > ‎Km

In this experiment we determined the Michaelis constant known as Km value that reflects the affinity of an enzyme for its substrate. A low value of Km indicates a high affinity of the enzyme for its substrate since maximum velocities will be attained at low substrate concentration. To determine the Km value we obtained the initial rates of enzyme-substrate interaction with double beam spectrophotometer. After that we used three types of plots that is based on the formula V = (Vmax x [S]) / ([S] + Km). These graphs were Michaelis-Menten, Lineweaver-Burk and Eadie-Hofstee plots. Since all of them are used for determination of Km value the results are slightly differs from each other. In our experiment Km values obtained from Michaelis-Menten, Lineweaver-Burk plots are said to be close to each other but the Km value obtained from Eadie-Hofstee plot is obviously different from the others. When we compare the R2 shows a huge difference compared to other plots and far from R2 value of 1. That’s why the Km value obtained from Eadie-Hofstee plot is unreliable. constants, which measures how well the data line up, Eadie-Hofstee plot

Laboratory‎ > ‎Analysis of Proteins

THEORY

Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. As a result they have different molecular structures, nutritional attributes and physiochemical properties. Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, as well as containing essential amino-acids, such as lysine, tryptophan, methionine, leucine, isoleucine and valine, which are essential to human health, but which the body cannot synthesize. Proteins are also the major structural components of many natural foods, often determining their overall texture, e.g., tenderness of meat or fish products. Isolated proteins are often used in foods as ingredients because of their unique functional properties, i.e., their ability to provide desirable appearance, texture or stability. Typically, proteins are used as gelling agents, emulsifiers, foaming agents and thickeners. Many food proteins are enzymes which are capable of enhancing the rate of certain biochemical reactions. These reactions can have either a favorable or detrimental effect on the overall properties of foods. Food analysts are interested in knowing the total concentration, type, molecular structure and functional properties of the proteins in foods.There are several methods for the determination of proteins in foods.

Kjeldahl method:

The Kjeldahl method was developed in 1883 by a brewer called Johann Kjeldahl. A food is digested with a strong acid so that it releases nitrogen which can be determined by a suitable titration technique. The amount of protein present is then calculated from the nitrogen concentration of the food. The same basic approach is still used today, although a number of improvements have been made to speed up the process and to obtain more accurate measurements. It is usually considered to be the standard method of determining protein concentration. Because the Kjeldahl method does not measure the protein content directly a conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration. A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of protein) is used for many applications, however, this is only an average value, and each protein has a different conversion factor depending on its amino-acid composition. The Kjeldahl method can conveniently be divided into three steps: digestion, neutralization and titration.

Principles

Digestion

The food sample to be analyzed is weighed into a digestion flask and then digested by heating it in the presence of sulfuric acid (an oxidizing agent which digests the food), anhydrous sodium sulfate (to speed up the reaction by raising the boiling point) and a catalyst, such as copper, selenium, titanium, or mercury (to speed up the reaction). Digestion converts any nitrogen in the food (other than that which is in the form of nitrates or nitrites) into ammonia, and other organic matter to C02 and H20. Ammonia gas is not liberated in an acid solution because the ammonia is in the form of the ammonium ion (NH4+) which binds to the sulfate ion (SO42-) and thus remains in solution:

N(food) ® (NH4)2SO4 (1)

Neutralization

After the digestion has been completed the digestion flask is connected to a recieving flask by a tube. The solution in the digestion flask is then made alkaline by addition of sodium hydroxide, which converts the ammonium sulfate into ammonia gas:

(NH4)2SO4 + 2 NaOH ® 2NH3 + 2H2O + Na2SO4 (2)

The ammonia gas that is formed is liberated from the solution and moves out of the digestion flask and into the receiving flask – which contains an excess of boric acid. The low pH of the solution in the receiving flask converts the ammonia gas into the ammonium ion, and simultaneously converts the boric acid to the borate ion:

NH3 + H3BO3 (boric acid) ® NH4+ + H2BO3- (borate ion) (3)

Titration

The nitrogen content is then estimated by titration of the ammonium borate formed with standard sulfuric or hydrochloric acid, using a suitable indicator to determine the end-point of the reaction.

H2BO3- + H+ ® H3BO3 (4)

The concentration of hydrogen ions (in moles) required to reach the end-point is equivalent to the concentration of nitrogen that was in the original food (Equation 3). The following equation can be used to determine the nitrogen concentration of a sample that weighs m grams using a xM HCl acid solution for the titration:

Where vs and vb are the titration volumes of the sample and blank, and 14g is the molecular weight of nitrogen N. A blank sample is usually ran at the same time as the material being analyzed to take into account any residual nitrogen which may be in the reagents used to carry out the analysis. Once the nitrogen content has been determined it is converted to a protein content using the appropriate conversion factor: %Protein = F´ %N.

Advantages 

The Kjeldahl method is widely used internationally and is still the standard method for comparison against all other methods. Its universality, high precision and good reproducibility have made it the major method for the estimation of protein in foods.

Disadvantages

It does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein. Different proteins need different correction factors because they have different amino acid sequences. The use of concentrated sulfuric acid at high temperatures poses a considerable hazard, as does the use of some of the possible catalysts The technique is time consuming to carry-out.

Enhanced Dumas method

General Principles:

A sample of known mass is combusted in a high temperature (about 900 oC) chamber in the presence of oxygen. This leads to the release of CO2, H2O and N2. The CO2 and H2O are removed by passing the gasses over special columns that absorb them. The nitrogen content is then measured by passing the remaining gasses through a column that has a thermal conductivity detector at the end. The column helps separate the nitrogen from any residual CO2 and H2O that may have remained in the gas stream. The instrument is calibrated by analyzing a material that is pure and has a known nitrogen concentration, such as EDTA (= 9.59%N). Thus the signal from the thermal conductivity detector can be converted into a nitrogen content. As with the Kjeldahl method it is necessary to convert the concentration of nitrogen in a sample to the protein content, using suitable conversion factors which depend on the precise amino acid sequence of the protein.

Advantages

It is much faster than the Kjeldahl method (under 4 minutes per measurement, compared to 1-2 hours for Kjeldahl). It doesn’t need toxic chemicals or catalysts. Many samples can be measured automatically. It is easy to use.

Disadvantages

High initial cost. It does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein. Different proteins need different correction factors because they have different amino acid sequences. The small sample size makes it difficult to obtain a representative sample.another protein determination methods are;

·        UV-visible spectroscopy

·        Biuret Method

·        Lowry Method

·        Dye binding methods

·        Turbimetric method

PURPOSE: 

In this experiment we determined theamount of protein in food product.for this purpose we used kjeldahl method.because,this method is most convenient and well accepted method.firstly,we converted the nitrogen to the ammonium bisulfate then ammonia was distilled with boric acid to form ammonium.In the titration step ammonium was titrated with hydrochloric acid.

PROCEDURE

·        2 gr of sample was taken into digestion flask

·        10 gr of potassium sulfate was added to increase the boiling point

·        catalyst which was cupper sulfate was added to the digestion flask

·        25 ml of sulphiric acid was added to the flask

·        then,digestion flask was placed on kjeldahl onit(adjusted to 400 °C for 40 min)

·        the digestion flask was heated until clear green color was observed

·        the digestion flask was placed into the machine.the distillation process was done by the machine

·        the ammonium borate solution was titrated with hydrochloric acid

MATERİALS

  • 2 gr sample

  • burette

  • potassium sulfate

  • erlenmeyer flask

  • copper sulfate

  • boric acid

  • sulphiric acid

  • HCl solution

  • digestion flask

  • NaOH solution

  • methyl red as indicator

  • Boiling chips

CALCULATION

We consumed 26,8 ml of hydrochloric acid in order to reach the end point for 2 gr sample

Amount of gr of NH3(gr)  = 26,8ml* 1,4015mg /1000 ml  = 0,0375 mg  = 0,0000375 gr

%protein = 0,0000375 /2 gr *100*6,25 = 0,011

DISCUSSION

In this experiment we determined the amoumt of protein for a given sample by determining the nitrogen content.ıt was found that the amount of proteıns in the sanple was 0,011%.this meant that 0,011% of 100 gr sample was protein.we used kjeldahl method for determining of protein. The Kjeldahl, Dumas and IR methods require very little sample preparation. After a representative sample of the food has been selected it can usually be tested directly. On the other hand, the various UV-visible methods require extensive sample preparation prior to analysis. The protein must be extracted from the food into a dilute transparent solution, which usually involves time consuming homogenization, solvent extraction, filtration and centrifugation procedures. In addition, it may be difficult to completely isolate some proteins from foods because they are strongly bound to other components.in the experiment we used potassium sulfate because the reaction needed high temperature to be completed so potassium sulfate increased the boiling point of the solution.

Laboratory‎ > Centrifugation

Centrifugation