SULPHYDRYL ANALYSIS

by Student’s name

Code+ course name

Professor’s name

University name

City, State

Date

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. This experiment introduces some methods for investigating and analyzing sulphydryl and disulphide groups in proteins.

Materials and methods

Measurement of Sulphide and Disulphide groups in a protein with DTNB

Lysozyme solution at a concentration of 0.5 mg/ml was used here. Four test tubes were set up and they were labeled as 1, 2, 3 and 4. 1.44 gms of urea were added into the test tubes of 2, 3 and 4. 0.1ml of 0.1M EDTA was added to the each tube.Afterward, 1.0 ml o f Lysozyme solution was added to test tubes of 1, 2 and 3 and shake well to mix. Then 50gms of Sodium Borohydride was weighed out and it was carefully dissolved in 2 ml of distilled water. Good care was taken when hydrogen gas was slowly evolved. 1 ml of Sodium Borohydride was added to test tube 2 and 1 ml of distilled water to test tubes of 1and 3. This increased the volume of each tube up to 3.0 ml by adding the water. One drop of Octyl alcohol was added to each tube, it was mixed well and shook to dissolve the urea. Tubes were placed in a water bath at 37°C and allowed the reduction reaction to proceed for 30 min. Then 0.5ml of 1M KH2PO4 which contained 0.2M Hydrochloric Acid was added to each tube. Wetted the sides of the tubes of destroy the Borohydride. 2 ml of acetone was added to each tube after 5 min mixed well. Then bubble nitrogen was passed through each tube for 2 min. 0.5ml of 10mM DTNB in 0.05M phosphate buffer pH 8 was added immediately after purging with nitrogen. Finally, bubble nitrogen was passed through each tube for 2 min to fill the gas space. Stopper the tubes and allowed to stand for 15 min at 37°C. 3ml glass cuvettes and Varian spectrophotometer were used for reading the absorbance at 412 nm. Then the instrument was zeroed on water. The tubes were taken straight from the water bath to the cuvette keeping the contents warm to prevent phase separation which will cause cloudiness and prevent a true absorbance reading.

Results

Table 1a of Lysozyme.

|Absorbance and Concentrations: Solutions |Absorbance (AU) |
|1 |0.065 |
|2 |0.928 |
|3 |0.116 |
|4 |0.095 |

Our data was faulty and thus could not be used for this calculation; this was due to errors in concentration of our protein samples. Hence we used other results that were more accurate.

Table 1b: Rectified, more accurate results

|Solutions |Absorbance (AU) |
|1 |0.185 |
|2 |0.193 |
|3 |0.104 |
|4 |0.089 |

Graphic Representations

[pic]

Graph 1: Standard curve of BSA .

From the table the absorbencies at 412nm of Tube 1, 2, 3 and with no proteins 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 tube 4 which had no protein. After subtraction, the absorbance values were 0.098, 0.473 and 0.011.

Consider Given the following:-
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. Given the formulae:- N = MW protein x absorbance x volume (6ml) 1200 X M (wt. Of protein in mg)
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 = 31.5X10-6 gm/ml

Molecular weight of the RNase = 12, 640.
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 = 2.5 X 10-9 X 6.022 X 1023 molecules/ml

Considering 4 disulphide groups per molecule of RNase,
Thus, No. of disulphide groups per molecule of RNase = 2.5 X10-9 X 6.022 X 1023 X 4 disulphide groups/ml
Therefore, Moles of disulphide groups = 2.5 X10-9 X 6.022 X 1023 X 4 /Avogadro’s number = 10 X 10-9 moles/ml Moles of disulphide groups = 10 nmoles/ ml

Determination of disulphide groups in Lysozyme using a Fluorimetric method

1ml solution of crystalline RNase was diluted in distilled water. This diluted solution was contained 31.5 microgms/ml of RNase of Molecular weight is 12,640. The mole of disulphide groups per ml of this solution was calculated. A standard curve of RNase solution using dilutions ranging from 1 to 10 nmoles disulphide per ml was prepared. And then all tubes were made to 1 ml. A solution without any disulphide was used as a blank. Including this blank there was needed 11 tubes. Duplicate tubes of 1:32 dilution of the lysozyme solution (0.5 mg/ml) were prepared. Diluted the 10-4 M fluorescein mercuric acetate (FMA) in 0.01M NaOH (solution provided) in the 1:10 dilutions with 1 M NaOH. 1 ml of this diluted Fluorescein mercuric acetate (FMA) is added to all test tubes. 8 ml of 1 M NaOH was added to all tubes and mixed well. Tubes were left to stand for 1 hour.

SPECTROFLUORIMETRY using the Cary Eclipse (back bench, sink end).

Spectrum of Fluorescein Mercuric Acetate (FMA)

Cary eclipse fluorescence spectrophotometer was switched on. Cary Eclipse software was selected and opened it for the various utilities to determine the spectral properties of the FMA so that optimum settings were used for disulphide assay and scan utility was selected and opened. The reagent blank of the standard curve tubes were taken and filled the cell about 2/3 full with carefully for not to any contamination. The outside of the sample cell was wiped gently with dry tissue and placed in the cell holder of the instrument. Then Pre-scan option was selected. Removed the popup window by clicking ok and started the scan which was allowed a minute to complete. The trace for excitation (red) and emission (black) were drawn on the graph window and values of the Excitation wavelength, Emission wavelength and the PMT (photomultiplier tube) voltage of the used instrument were recorded. The graph was saved in the word file.

Spectrum of Fluorescein Mercuric Acetate (FMA) The spectral properties of the FMA was determined to obtain the optimum settings for the disulphide assay.

[pic]

Graph 2: Graph obtained in Cary eclipse Fluorescence Spectrophotometer showing the spectrum of Fluorescein Mercuric Acetate (FMA).

Disulphide Fluorescence Quenching Assay
The Simple Reads utility was opened and went to Setup at left top and changed the Excitation (Ex) and Emission (Em) wavelength setting to the optimum settings. The slit width was left at 5 nm. Then went to the Options tab of the popup window and selected Manual for the PMT voltage and set the voltage at the value that was obtained in the scan. OK was pressed to effect this change. After PMT settings were completed, the fluorescence reading into the onscreen report was recorded by pressing the read at top of screen. This was the zero disulphide (blank). Then the relative fluorescence of each tube of the standard curve and the duplicate Lysozyme sample were READ with the same settings by carefully replacing the sample in the sample cell. After completion of readings washed out the cell with water thoroughly and dried carefully. Disulphides quench the fluorescence of FMA in alkaline solution so the protein standards and samples had less fluorescence than the blank. A graph was plotted between the fluorescence reading and against the number of disulphide groups (in nmole) for the RNase standards. From the fluorescence reading of Lysozyme on the standard curve was worked back via dilution factors to the number of moles of disulphide groups per ml of original solution. The number of moles of disulphide groups per mole of protein was calculated.

The relative fluorescence of all the 11 tubes of the standard curve and the 2 tubes with the duplicate lysozyme sample were read using the spectrum settings and the PMT voltage was taken in such a way that the scale was between 800-1000 relative fluorescent units. The result was obtained as follows.

Instrument Parameters

|Instrument Parameters Instrument |Cary Eclipse |
|Instrument Serial Number |EL04094118 |
|Data mode |Fluorescence |
|Ex. Wavelength (nm) |500.00 |
|Em. Wavelength (nm) |522.98 |
|Ex. Slit (nm) |5 |
|Em. Slit (nm) |5 |
|Ave Time (sec) |0.1000 |
|Excitation filter |Auto |
|Emission filter |Open |
|PMT Voltage (V) |554 |

Results Flags Legend

@ = Over-range

|Tubes |Read Int. (a.u.) |
|1 |833.928 |
|2 |834.618 |
|3 |833.477 |
|4 |730.366 |
|5 |619.217 |
|6 |421.790 |
|7 |451.595 |
|8 |349.788 |
|9 |266.642 |
|10 |223.924 |
|11 |151.829 |
|12 |4.625 |
|13 |4.289 |

A graph was plotted between the obtained fluorescence readings against the number of disulphide groups (in nmole) for the RNase standards.

[pic]

Graph 3: Shown are No. of disulphide in RNase solution on x-axis and Fluorescence readings on y- axis.

From the graph, the corresponding values for the absorbance of the duplicate Lysozyme solutions are 10.4 nmol/ml when converted comes to 10.4 X 10-3 μmol/ml, the duplicate Lysozyme solution was prepared in the 1:32 ratio, and volume of the lysozyme was is 31.25 μl, The concentration of the lysozyme from stock was 0.5 mg/ml, thus,

The weight of the lysozyme in the 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 = 15.625X10-6 gm/13,930

= 1.12167 X 10-9 mol = 1.12167 nmol

Therefore, The number of moles of disulphide groups in lysozyme solution one = 10.4 nmoles /ml.

Discussion

From the table 1b, Lysozyme activities of four samples 1, 2, 3, 4 were 38U, 21U, 20U, 19U respectively. The specific activities of four samples 1, 2, 3, 4 were 51.3, 24.27, 12.46, 12.3U/mg respectively. But the specific activity values could not give expectable results. From the observation of these values, the values are decreased progressively. The total activities of these four samples Lys 1, 2, 3, 4 are 235.6, 130.2, 124, 117.8U respectively. These values are increased progressively. The Total activity values could have given the good and expectable results. From the analysis of measurement of sulphide and disulphide groups in a protein with DTNB, tube 2 contained the urea and sodium borohydrate, tube 1 did not contain urea and sodium borohydrate. From the table 3, tube 1 had 1.365 sulphide groups and tube 2 had 6.588 sulphide groups in their protein sample. When a protein is partially denatured, that means only part of it is converted into a form insoluble under conditions under which the native protein is soluble, the insoluble fraction has the number of reactive SH and S-S groups characteristic of completely denatured protein, whereas the soluble fraction has the number characteristic of protein which has not been denatured at all. Finally, when a protein is converted by urea into a form which has an increased number of S-S groups, that form is insoluble in a medium in which native protein is soluble. In denaturation, formation of insoluble protein and increase in detectable SH and S-S groups are closely related aspect. From that tube 2 had increased sulphide groups than tube 1. Coming to Sodium borohydride (NaBH4), it can be destroyed by acidification or with acetone. Reduction of the --SS-- linkage in pure chemical compounds with this agent Acetone alone destroyed the excess NaBH4, but after treatment with DTNB a yellow colour developed slowly even in solutions free of --SH groups, and absorbance of solutions with --SH present gradually increased with time For this reason a mixture of HCl with acetone was adopted. Acetone also served as an antifoam agent, but too much acetone precipitated protein which no longer dissolved at neutrality.

From the table 3, the no. of sulphide groups in tube 2, are 6.588 moles and the no. of sulphide groups in tube 3 are 0.158 moles. So tube 2 which had sodium borohydrate has increased sulphide groups because it reduction the sulphide linkage and encourage to precipitate the protein. That’s why the protein insoluble which contain increased sulphide groups. Where as tube 3 which had no sodium borohydrate has decreased sulphide groups when compare with the tube 2. From the graph 3, which was shown the no. of disulphide groups in RNase solution on x-axis and Fluorescence readings on y- axis, the mole of disulphide groups per ml of protein is 10.4 nmoles which is obtained from the fluorimetric method.

Conclusion

The sulphide groups in the protein with DTNB in the tubes of 1, 2 and 3 having 1.365moles, 6.588moles and 0.158 moles respectively. There is much difference in number of free Sulphide groups in tubes 1 and 2 will be 5.223moles. So the tube which has urea has more sulphide groups than other. At the same time and similarly there is much difference in number of free sulphide groups in tubes 2 and 3 will be 6.430. So the tube which has sodium borohydrate has increased sulphide groups than other tube. From the second technique i.e by disulphide fluorescence quenching assay gives the number of moles of dilsulphide groups per mole of protein of the lysozyme was 10.4nmoles

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