Introduction
The purpose of the experiment was to test the free sulphydryl groups that may be important for the enzyme activities as well as the roles that the disulphide bonds plays n the stabilizing of the structure that is 3 dimensional of the enzymes and proteins. The latter purpose is vital in the study of proteins. The results for tube 2 showed that N= 6.588 and that tube 3 showed that N= 0.153 sulphide groups in the samples. tube 2 contained Urea and sodium borohydrate. When the proteins were denatured partially, it meant that only part of it was converted to a form that is insoluble under the conditions which the native proteins are soluble. The insoluble fractions had the number of reactive SH as well as the S-S groups characteristics of the completely denatured proteins.
Materials and methods
The concentration of the proteins determines the likelihood of formation of gel below certain levels comes to variation as per the protein utilization which means that gelation will not take place. With low levels of proteins, the protein network becomes difficult in establishment which as a result leads to the conclusion that the interactions between proteins tends to occur within the molecules and that a gel framework cannot be established due to the fact that with the increases in protein contents which tends to strengthen the texture of the resultant firer gel as water gets tightly bond with the proteins.
Cysteine is of vital importance as an amino acid and the free sulphydryl groups may also prove to be of importance in the enzyme activities whereas the disulphide bonds plays important roles in the stabilizing of the 3 structure dimension of the enzymes as well as proteins. There have been a couple of methods that have been developed for the purpose of estimation of the sulphydryl group whereas the Disulphide groups are measured usually by the modification of one of the methods after the proteins have been reduced. The purpose of the experiment is to introduce some of the methods of investigating as well as analysing sulphydryl and the disulphide groups in the proteins.
The reagents that are used in the experiment include crystalline urea- tray,
Octylalcohol -fume cupboard due to the fact that it is toxic, Sodium borohydride –tray, Acetone (fume cupboard) due to its toxic nature as well as the toxicity of the reaction. The stock solutions that are used are: 0.1M of Ethylenediaminetetraacetic acid- EDTA in the fridge, 1 molar of Monopotassium phosphate- KH2PO4 in a 0.2 molar solution of Hydrochroloric acid in the tray and one molar solution of sodium hydroxide-NaOH. The protein solutions that have been used are Lysozyme provided at 0.5mg/ml and Ribonuclease (RNase) in concentrations of 1mg/ml.
Due to the fact that there were some errors in our experiment, we had to rely on someone else’s findings so as to get the results due to some errors that arose which as one of the results meant that there were some negative errors in the findings.
Determination of disulphide groups in lysozyme using a fluorometric method
With the solution of crystalline being diluted in the distilled water which posses 31.5 μg/ml of RNase, with the molecular weight being at 12,640. The moles of the Disulphide groups per ml of the solution through the use dilutions that range from 1-10 n moles of the disulphide per every ml of the solution that was prepared. The tube that did not have any disulphide was marked as blank which needed 11 tubes. Duplicate tubes that were of of 1:32 dilution of the lysozyme solution (0.5 mg/ml) were prepared with the tubes being made to 1ml with all the tubes that did not contain disulphide being labelled as blank. The duplicate tubes of the 1:32 dilution of lysozyme solution were prepared. fluorescein mercuric acetate (FMA) diluted to 0.00001m in the 0.01m NaOH in 1:10 dilutions with the 1M NaOH. Diluted Flouorescein mercuri acetate in 1ml was added to all of the test tubes with 8ml of the 1M NaOH being added to all the test tubes and mixed well with the tubes being left for one hour.
Protein molecules that posses the same primary structure normally all posses the same secondary and tertiary structures the reason for the is that protein orient is such a certain way has to do broadly with integrations of each of the particular kinds of amino acids. The polar charged amino acids are fully charged which means that they have acidic or even basic components in their side chains which then dissociate under physiologic conditions which leaves a full charge. The side chains readily form bonds that are ionic with possession of other full charges which makes them very reactive.
Results
The lysozyme solution is used at a concentration of 0.5mg/ml with 4 test tubes set up with labels 1-4 which contain 1.44 grams of urea after which 0.1 M EDTA was added to each tube. An addition of lysozyme is added to each of the tubes in quantities of 1.0ml in the the tubes 1-3. 50 mg of sodium borohydride were ten weighed and carefully dissolved in 2 ml of the distilled water. Care in the latter should be taken due to the fact that hydrogen is slowly evolved. An additional 1 ml of sodium borohydride is put in to the second tube with 1 ml of the distilled water to each of the tubes that were labelled 1 and Water was then added so as to bring up the volume to 3.0 ml in each of the tubes with one drop of oytyl alcohol to each of the tubes. The mixtures were then shaked well so as to dissolve the urea. The next step was the placement of the placement of the tubes in a water bath at 37 degrees Celsius so as to allow reduction reaction for a period of 30 minutes. 0.5 ml of 1 molar of KH2PO4 which contains 0.2 molar of HCL was added to each tube with the sides of the tubes wet which was for the purpose of destroying the borohydride. After five minutes of waiting, 2 ml of acetone was added to each of the tubes for a time period of 2 minutes to each of the tubes. After the two minutes elapsed, nitrogen was bubbled through each tube for a time period of two minutes. Immediately after pouring the Nitrogen 0.5ml of 10 molar DTNB in the 0.05 M phosphate buffered of p 8 was added so as to make the total volume in the tube to contain 6.0ml with water. In the final step, nitrogen was bubbled through each of the tubes for 2 minutes so as to fill the gas spaces. The tubes were Stoppard so as to allow them to stand for 15 minutes at the 37 degree Celsius temperatures.
Tube 1 2 3 4
Absorbance 0.065 0.928 0.116 0.095
The measure of the quantity of light that was absorbed by the sample- absorbance in the results showed that in the first tube, the rate was 0.065 units, 0.928 units in the second tube, 0.116 units in the third tube and 0.095 units in the forth tube. The ex wavelength was highest at 500 nm.
Calculations
Weight of lysozyme in 1000μl lysozyme solution = (31.25 μl/1000 μl) X 500 μg/ml = 15.625 μg/ml
Therefore, No. of Moles of lysozyme = Weight/Molecular weight = 15.625X10-6gm/13,930 = 1.12167 X 10-9 mol = 1.12167 nmol
Therefore: number of moles of disulphide groups per mole of protein
= 10.4 n moles /ml.
TUBE 4
The absorbencies at 412nm of Tube 1, 2, 3 and blank (no
protein) were 0.270, 0.655, 0.193 and 0.182 respectively.
Absorbance values of the tubes of 1, 2 and 3 were subtracted
From the 4th tube (blank). After subtraction, the absorbance
Values were 0.098, 0.473 and 0.011.
Molar absorptivity is 12,000/ M, cm.
Molecular weight of the lysozyme is 13, 930.
Weight of protein (M) is 0.5 mg from the given data.
Volume is 6 ml.
N= MW protein x absorbance x volume (6ml)
1200 X M (wt. Of protein in mg)
The number of sulphyde groups (N) in each tube was
Calculated from this formula
N=
For Tube 1: N = 1.365.
Similarly,
For TUBE 2: N= 6.588.
For TUBE 3: N= 0.153.
Calculation of moles of disulphide groups per ml
The weight of RNase in the 1 ml of diluted solution = 31.5 X 10-6 gm/ml. Molecular weight of the RNase= 12, 640. Formula for number of moles: No. of moles= Weight/ Molecular weight. Number of moles of RNase in 1ml = 31.5X10-6/12,640 =2.5 X 10-9 moles/ml No. of molecules of RNase per ml =No. of moles of RNase X Avogadro’s number (N) =2.5X10-9 X 6.022 X 1023 molecules/ml There are 4 disulphide groups per molecule of RNase, Hence, No. of disulphide groups per molecule of RNase =2.5X10-9 X 6.022 X 1023 X4 disulphide groups/ml Therefore, Moles of disulphide groups = 2.5X10-9 X 6.022 X 1023 X4 /Avogadro’s number = 10X10-9 moles/ml Moles of disulphide groups = 10 nmoles/ ml.
The numbers of Disulphide in the RNase solution on the X axis against the Fluorescence reading from the graph shows that the corresponding values for absorbance duplicate the lysozyme solutions.
Proteins contain potential sulhydryl groups which are vital in the processes of cellular respirations. One of the ways of altering many of the proteins is through the dissolving of them in urea or amide solutions. When a protein gets denatured-partially, it means that some part of it is transformed into forms that are insoluble under the conditions which make the native protein soluble which therefore means that the insoluble fraction contains the reactive SH as well as S-S characteristics of the group that is completely denatured. When a protein is converted by Urea into a form that has a number of increased S-S groups, the form makes some sort of medium that is insoluble through which the native protein is soluble. In the denaturation process, the creation of insoluble proteins as well as increases in detectable SH and S-S groups are aspects that are closely related. Te groups of sulphide that was in the proteins that are closely related with DTNB in the tubes containing 0.1 M (Sen, 2000, p 448).
The methods of analysis of the protein sulfhydryl (SH) group in the food systems has been largely overlooked at despite the changes in the SH group concentration affecting both the physical as well as the nutritional characteristics of the high protein foods. Food scientists s well as technologists require improve understandings of the protein SH chemistry so as to design the processes that minimize the losses of the thiol groups.
Discussion
As of the results one of the things that can be determined is that Sulphydryls plays an important role in the roles of protein functioning as well as folding. The sequence of the amino acid of the protein determines the native conformation. The protein molecule tends to fold impulsively during or even after the biosynthesis process. The process depends heavily on solvent water as the results show which can also be a lipid bi-layer, the pH, the concentration of salts as well as well as the temparatures-37 degrees Celsius and the possibility of co factors presence in the solutions which are all depicted in the varied results that were carried out.
The minimizing of the numbers of hydrophobic side chains that is exposed to water is one of the major vital driving forces that lay behind the folding process. The formation of the intermolecular hydrogen bonds makes another vital contribution to the sustainability of the stability of the proteins. It was now on a factual basis to establish that the strength of the hydrogen largely depends on the surrounding environment. the latter is due to the fact that the hydrogen bond tends to get inserted in a hydrophobic core that contributes more than the H-bonds that are exposed to the hydrous environment up to stability of the innate state.
The molecules of proteins may contain crosslink’s of a non covalent such as salt bridges or covalent such as disulphide bonds. The crosslink’s tend to lower the conformational entropy of the molecule which was compensated for by the decline in the binding energy. The presence of the crosslink’s is a great addition to stability of the protein’s structure that is native which therefore makes the molecules to become resistant to the unfolding or even the denaturation. Denaturation however is less likely to be one of the driving forces due to the fact that one of its main driving forces include entropy that is conformational which in this case is greatly reduced (Manual 2013).
Some amino acids bond more with water than others which is the reason that some proteins contain large amounts of amino acids that tend to bind molecules of water that are large. Some proteins need the presence of some amounts of electrolytes so as to enable them to be soluble. The ions that interact with the charged group on the protein surface as well as with the water present which in the long run tends to increase the amount of the water bond to the protein.
Conclusion
Sulphide groups that were in the proteins that had DTNB in the tube 1,2 and 3 show much difference in the numbers of free groups than the others which at the same time similarly showed that there is much difference in the numbers of free sulphides in the 1 and 2 tubes. This leads to the tubes that have urea containing more sulphide groups than each other which at the sam time meant that there is much difference in the numbers of sulphide groups in the tubes 2 as well as 3 which meant that the tube that had sodium boroydrate had increased had increased the sulphide groups in the other tube.
Reference List
http://www.ijitee.org/attachments/File/v2i3/C0473022313.pdf
http://bbs.scu.edu.cn/wForum/bbscon.php?bid=153&id=9151&ap=547
http://www.piercenet.com/instructions/2160311.pdf
SEN, C K, PACKER, L, & HÄNNINEN, O. (2000), Handbook of oxidants and antioxidants in exercise, Amsterdam, Elsevier, http://site.ebrary.com/id/10204235.
