Expt.

03: Fluorimetric Studies of Protein Folding using Light Scattering

THEORY:

Proteins are large macromolecules consisting of a single or multiple chains of amino acids.
Proteins have a diverse functional role to play in biological systems , ranging from physical
support, to pH regulation, to nutrient transport, to regulation of entry of substances in and out of
the cell, to cellular compartmentalization, cell communication, signaling, metabolic inter-
conversion, enzymatic catalysis and many other functions. As such, protein function is almost
wholly dependent on its structure. Protein structure
occurs at four levels of organization, and the process
by which the protein moves from a lower order of
assembly to a higher order of assembly, forming a
more stable and functional molecule, is known as
protein folding. It is a cooperative process and goes
through several energetic intermediates, now known
as molten globule states, and by using denaturing
agents such as urea we can disrupt this folding
process at different stages, obtaining intermediates
with differing degrees of aggregation. The aggregates
cause an increase in scattering, which can be
observed fluorimetrically. The protein that we have Fig. 1 Ribbon Diagram of BSA, showing -helices
worked with in this experiment is bovine serum and strands
albumin (BSA).
Denaturation of a protein:

Denaturation is a process wherein a protein loses its tertiary structure and unfolds, under the
influence of a chemical reagent that disrupts the bonding forces that stabilise its tertiary
structure, or under the influence of a physical agent like heat. Chemical reagents that destabilise
the bonding interactions that hold a proteins tertiary structure together are known as
denaturants. To understand how a denaturant works, we need to remember the forces that
stabilise the tertiary structure of a protein. These are:-

a. Hydrogen bonding;

b. Hydrophobic interactions;

c. Disulfide linkages;

d. Van der Waals interactions;

e. London forces;

f. Dipole-induced-dipole interactions.
Hydrogen bonding is perhaps the most important of the non-covalent interactions that stabilise
the tertiary structure and the native folded state of the protein. Hydrogen bonding requires that a
hydrogen atom be bonded to an electronegative atom and be held in the proximity of another
electronegative element. For a protein, the N-H bond interacts with a carbonyl O atom on a
different amino acid residue that is spatially proximal to it, but not necessarily close to it in the
amino acid sequence of the protein. Urea is an extremely good denaturing agent, as it does not
chemically affect the protein sequence but simply dissociates the hydrogen-bonding interactions
that stabilise the tertiary structure of the protein. It does so because of its structure-dependent
chemical properties - hydrogen bonding occurs when a hydrogen atom is proximal to two
electronegative elements, to one of which it is bonded. This causes the electron distribution
across the X-H bond (where X refers to an electronegative heteroatom typically N or O) to be
perturbed and drawn slightly to the other, non-bonded electronegative atom. This is why
hydrogen bonding is classified under non-bonding interactions. The electronegative atom to
which H is bonded serves as the H-bond donor, while the other serves as a H-bond acceptor. In
case of urea (NH2CONH2), the nitrogen atoms can serve as excellent H-bond donors, and the
carbonyl O atom can serve as an excellent H-bond acceptor. Thus, urea substitutes the H-
bonding function performed by the amino acid side chain residues on a particular secondary
structural motif and as a result, the tertiary structure disassembles, resulting in secondary
structural aggregates that led to an increase in scattering. Thus, differing concentrations of urea
were used with a standard BSA solution and the increase in scattering was plotted
fluorimetrically.

The mode of scattering that we have studied is Rayleigh scattering, which is elastic in nature,
and as a result, excitation of the sample and fluorimetric analysis was conducted at the same
wavelength (400 nm). This is a deviation from standard fluorimetric protocol, which typically
excites the sample at a lower wavelength and then observes the fluorescence spectra over a
range of wavelengths greater than the wavelength at which excitation took place. However,
since those relaxations commonly observed as fluorescence are non-elastic, in this experiment
we are not concerned with them, and analysis of intensity was observed over a time period at
400 nm for samples containing different concentrations of urea.

Materials and Methods:

1. A stock solution of 2M BSA was prepared by dissolving 13.2 gm of BSA in 10 ml of
deionised, MiliQ water.
2. A stock solution of 6M urea was prepared by dissolving 3.6 gm of urea in 10 ml of deionised
MiliQ water. This stock solution was later used to prepare auxiliary urea solutions at
different concentrations by dilution.
3. The solutions for the experiment were prepared according to the following protocol:
 Concentration Volume of 6M Volume of Total volume Volume of Total Volume of Urea
of auxiliary urea stock MiliQ of urea 2M BSA + BSA solution in
urea solution solution taken water solution (ml) stock soln. cuvette (ml)
(M) (l) added (l) added (ml)

1M 170 830 1 1 2

2M 340 660 1 1 2

3M 510 490 1 1 2

4M 680 320 1 1 2

5M 850 150 1 1 2

6M 1000 0 1 1 2

4. The data and graphs for the series of samples were taken and plotted using Origin.
DATA:

1. Intensities of various solutions containing different concentrations of urea over time.

bsa + 1M bsa + 2M bsa + 3M bsa + 4M bsa + 5M bsa + 6M
bsa urea Urea Urea Urea Urea Urea
Intensity Intensity Intensity Intensity Intensity Intensity Intensity
Time (min) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.)
0.000833333 201.139389 260.3234558 305.3018799 351.3450317 414.093689 457.5618896 491.4267273
0.0025 202.6969604 259.8105469 305.3143005 353.3662109 412.3368835 456.3876038 491.9144287
0.004166667 202.7420044 261.6508484 306.4279785 351.3167114 411.7371216 457.0776367 494.2133789
0.005833333 200.6120605 258.2351379 304.2658081 351.8727722 412.4710999 459.162262 493.4630432
0.0075 202.7211761 262.0347595 306.4725037 352.6811829 413.5436401 455.9938049 489.9673157
0.009166667 202.0128632 260.331543 305.9037476 353.4293823 414.6975708 454.5186157 491.5422974
0.010833334 202.0662231 259.6594543 305.3958435 349.3800354 414.0736084 454.4225159 490.7281494
0.0125 201.6540222 260.1183777 305.7466736 351.4007263 414.3683167 455.5878296 489.6155396
0.014166667 202.1841431 259.539917 305.8686523 352.4663696 412.8253784 452.9294434 492.3802185
0.015833333 204.4755402 259.1003723 304.5340881 355.0724792 413.6961365 450.6290894 493.7558899
0.017500002 201.1882172 260.4703674 305.6871948 351.5028381 415.6604309 451.9474792 493.274353
0.019166667 203.522583 259.0557251 305.4290771 352.6552429 415.3739929 452.3797913 490.3878174
0.020833334 204.6779938 259.1860046 304.9844055 352.2425537 416.4277344 452.5510864 489.2123108
0.022500001 202.3846283 261.2542419 305.552887 355.3310242 413.2606812 450.5970154 488.9832153
0.024166668 201.775177 259.7471008 307.0708618 352.6278076 417.1347046 453.7600403 489.7767334
0.025833335 202.4144592 259.6162109 304.5428467 355.0368958 415.8242188 454.3059387 492.9257507
0.0275 203.2194214 259.6932068 303.8369446 353.8226929 414.2145996 452.7698364 483.9862366
0.029166667 204.0570679 260.15625 306.8944092 354.6746216 414.4657288 453.1448059 490.2322388
0.030833334 204.529068 256.8418579 304.3221741 355.6875305 415.6950989 452.9353027 488.0030212
0.032500003 203.9811401 259.3648987 306.2705078 353.6634216 416.8735962 455.7192993 489.6845093
0.034166668 199.816391 258.1882324 304.5520935 356.4785461 415.7110596 448.8518372 489.9977112
0.035833336 201.4517822 258.039978 306.5588684 355.7807617 419.1058655 455.5481873 490.691925
0.037500001 204.9482422 256.6102295 305.7565918 356.9191284 411.0007935 450.3423767 484.4204102
0.03916667 203.1222839 257.721405 306.2047119 354.2267151 413.6811829 456.7573853 494.883606
0.040833335 202.3907623 259.2283936 303.4484253 351.6199341 413.3717346 452.8149109 493.5542908
0.0425 204.9600372 259.2099609 307.0698242 354.1289063 413.3495178 454.5454712 491.1654053
0.044166669 202.7451019 258.557373 304.6818542 354.959259 416.706604 451.3452148 492.761261
0.045833334 200.0073242 258.7855225 303.9818726 353.5868835 408.9620667 453.8176575 486.1535034
0.047500003 200.0983582 260.0189819 304.4793091 354.0276184 415.3609314 454.8215942 487.7081909
0.049166668 200.6949463 258.5514526 304.2418213 354.9919434 413.8767395 456.8783264 487.9621277
0.050833333 201.1761169 255.4279327 304.85672 350.0299377 413.1227112 457.9440308 490.7557983
0.052500002 201.9020691 254.6516724 304.4778137 351.7385559 414.163147 456.6112366 486.1413879
0.054166667 202.3219757 254.0640869 304.113678 352.3362122 413.2950745 459.3160706 488.1176758
0.055833336 202.5325775 255.2804871 298.5080261 351.9056396 417.6290588 460.8449402 485.5082397
0.057500001 202.4028168 256.2163696 304.634491 352.6747437 408.7948303 455.449585 481.4067383
0.059166666 200.6236572 253.5648651 301.8172302 352.1511841 415.7468872 463.1815186 492.5796814
0.060833335 201.8212891 256.4665527 300.2268677 352.3284607 416.634491 454.8305359 488.4913635
0.0625 200.8999939 255.7749176 301.3856506 352.5044861 420.6228027 464.5053101 486.9282227
0.064166673 203.2069702 255.1342621 301.0271301 354.8960571 416.8455811 463.9631958 483.9364014
0.065833338 202.3473053 254.0471497 300.7903137 351.7044373 417.4008789 462.8742981 470.9321289
0.067500003 202.2025909 255.6820221 300.008606 352.1766052 416.2983093 462.6880798 483.7851563
0.069166668 201.5782166 255.6827393 301.4345398 354.3786316 415.0759888 464.0534973 478.0677795
0.070833333 202.9665375 254.3529816 299.0834961 351.9083862 415.0778809 467.6091309 488.6976318
0.072499998 201.7148895 257.0082092 298.51651 353.2843628 415.0838318 471.5213318 475.6832886
0.07416667 203.4076233 256.8905334 300.3370972 353.2327881 413.2635193 472.7268066 482.5211182
0.075833336 200.3502045 253.9291382 299.7782898 353.1906128 417.5127563 473.2643433 482.3967285
0.077500001 201.7086792 257.7958069 299.7849731 348.665802 419.4240723 486.3894043 483.8336792
0.079166666 202.5601654 255.0167694 301.1995239 352.3271484 416.0480652 477.5447693 480.5223694
0.080833331 200.2619171 256.4169617 302.1926575 351.5445557 417.9432068 482.0176392 485.2171631
0.082500003 199.9877319 257.1680603 299.6250916 353.653717 422.2233276 484.9249878 482.0141296
0.084166668 201.3305511 257.4533691 300.0194702 351.9100342 420.633606 481.7390747 479.9529114
0.085833333 201.0943756 257.5727539 296.4496765 351.9676208 419.4839783 479.8432312 480.3284607
0.087499999 198.9543762 257.0299072 298.4856873 353.0884399 416.6954041 479.0802917 483.6104126
0.089166664 202.1257019 256.7852783 298.5856018 354.6729736 419.286499 483.7449341 483.993988
0.090833336 202.8712463 255.8562469 299.0986328 352.6347961 417.9390564 473.1864929 480.5610962
0.092500001 202.9429474 256.7789612 299.5926819 352.5176392 419.4787292 475.8408203 471.1175537
0.094166666 203.9508209 256.3009033 298.4064636 353.1065979 424.5823364 476.8646851 482.2834167
0.095833331 202.0703888 257.5429077 298.7713623 354.2610168 413.6561279 479.29953 479.8200989
0.097499996 204.809494 256.5816956 300.5974426 351.4985046 419.7518005 470.0496826 483.8797302
0.099166669 203.5099335 256.5931091 299.0163269 354.1037292 418.314209 462.8500061 483.8872986
0.100833334 204.9248962 255.8184814 297.8987732 353.3418884 410.1388245 461.3635864 476.7283936
0.102499999 202.0337219 256.5039673 300.100647 353.5839539 421.9181213 458.6468811 481.6026306
0.104166664 204.3491974 255.731308 300.4288025 354.5965881 416.830658 452.480835 483.1036682
0.105833329 202.5045166 258.8806152 301.6902466 350.6018677 421.4035339 455.0325012 480.7253113
0.107500002 204.1741028 255.7419891 297.5699768 352.801178 422.8779907 450.051239 483.9950867
0.109166667 204.1191406 254.4447174 299.991333 349.7927246 425.2500916 448.7357788 482.4250488
0.110833332 201.9337769 257.8442383 300.1307983 352.4679565 419.8383789 449.026886 484.4621582
0.112499997 204.9921722 257.6278687 297.5324707 353.6496887 424.3817139 449.2667236 485.5196533
0.114166662 200.6316071 255.1173553 297.2728882 353.7148132 424.1101074 451.3722229 481.5389404
0.115833335 204.1627808 256.3078003 298.9737854 353.0561523 423.7666016 446.4125061 479.7562866
0.1175 202.3937378 256.0643311 297.7348022 353.781311 421.6778259 447.7799988 480.509613
0.119166665 202.0083313 258.8305359 297.1796265 351.8989258 425.5908508 445.0801086 478.8541565
0.12083333 202.1270142 256.1701965 298.8803406 352.151947 422.8199463 450.284668 478.5405884
0.122499995 204.8699646 255.0947418 295.4502258 352.1429443 424.6988525 450.3396606 482.2695007
0.124166675 202.3270721 258.2168884 297.5392151 351.6357727 425.0163269 453.5195923 482.2558899
0.125833333 202.396225 258.6219482 299.2040405 352.7468872 426.2704163 449.0635681 481.4211426
0.127499998 202.5469971 258.1732788 296.2765198 354.0271606 422.9423828 450.2387085 477.1413574
0.129166663 203.3039703 257.6016541 296.1295166 354.553009 429.4367065 450.8419189 483.1997681
0.130833328 202.7091522 256.3102417 298.0624695 353.6381836 426.5852051 448.0865784 482.3392639
0.132500008 203.1242371 258.0466003 297.5610046 351.7322388 419.7470703 443.3917847 482.8409119
0.134166673 204.3324738 257.7608948 294.8978271 350.9552002 424.9068604 450.0708008 478.071106
0.135833338 202.4426727 257.9330139 296.4527283 354.6663513 422.4155273 451.6663208 480.5505066
0.137500003 202.1621246 256.3412781 292.3369446 353.0296631 425.7839355 457.9089355 480.8459778
0.139166668 203.6694336 258.124939 294.1350403 355.9533997 424.4515686 453.8764648 484.7624207
0.140833333 205.9407349 257.1421814 296.5899658 354.1244507 423.6675415 454.2626648 484.6116028
0.142499998 202.1011505 258.5392761 297.1154785 353.9820862 419.2070313 450.2483215 485.0068665
0.144166678 203.3820343 257.24823 299.0493164 355.9666138 424.5910034 451.1385498 479.1269836
0.145833328 203.8631287 257.2202454 296.9927979 352.7349548 425.1841431 455.3628235 481.6105347
0.147500008 204.2390442 257.9514465 298.5203247 354.2835693 423.2698975 453.9034729 481.4160767
0.149166659 203.4278717 257.0299683 298.6975708 355.1289673 424.8225098 452.3422546 483.8311768
0.150833339 201.487915 256.0778198 290.663269 354.6239319 424.3250427 456.0587463 481.940094
0.152500004 203.4983826 256.4622498 295.8473206 352.7297363 423.5909729 455.3332214 481.9377136
0.154166669 202.8598022 258.0601196 296.6524658 355.2685242 422.0791321 452.3060303 482.9554138
0.155833334 201.8114777 254.8210602 298.7588196 354.4387817 413.7254639 454.6578674 483.5725098
0.157499999 202.3786011 254.0967102 294.8576355 352.8085632 421.5736389 453.5358582 485.0762634
0.159166664 204.8556213 254.4004974 297.1468506 355.9074097 422.765625 454.5803528 482.7515564
0.160833344 202.5010986 255.8743286 301.9699402 356.4195557 425.0108337 452.0338135 481.612854
0.162499994 201.93396 255.2838593 297.8619995 355.6220398 422.7485657 454.6603699 483.394165
0.164166674 203.5562592 257.6567383 297.9095764 353.6125488 420.8679504 451.6463623 483.3491821
0.165833324 203.1757202 257.7960815 297.9559631 353.6030884 421.5722656 452.2007141 476.5784607
0.167500004 205.5727081 256.8013611 299.2048645 352.9567566 424.4048157 452.782959 485.5947571
0.169166669 204.294693 256.2400818 299.4231262 351.7470093 420.5865173 451.5232849 485.8843384
0.170833334 202.5651245 259.0365906 302.6826172 354.4932861 426.0808716 452.3673096 484.2144165
0.172499999 202.7561188 257.3784485 301.3575745 353.7329102 421.2486572 451.0602417 484.0699463
0.174166665 203.7331696 257.2128296 301.8911133 350.631073 421.7265625 451.0596008 486.4433594
0.17583333 203.7550049 255.2280426 300.1770325 352.2390137 422.4530334 450.3177185 483.0056763
0.17750001 203.4040833 256.2640991 302.885498 351.5101013 423.2502747 449.7608948 481.9959717
0.17916666 202.9538727 254.1962891 305.3886719 350.9850769 422.5601501 452.6034546 482.6361084
0.18083334 202.7359619 255.6233673 305.4711304 353.0185852 421.8288879 450.7770386 484.3529663
0.18249999 203.7068329 255.8231354 307.3095703 349.4362793 425.0205994 452.401886 485.8811646
0.18416667 203.2382202 256.7260742 306.5163574 351.2441711 421.3686829 449.4703369 483.5952454
0.18583335 202.4248047 257.4997559 307.3759155 350.0137939 424.7409363 454.2617493 486.3781738
0.1875 201.144577 255.2258606 311.9224854 350.9543457 422.4951477 449.2876587 485.412384
0.18916668 202.6526184 256.2469177 313.0081787 351.2421265 417.8114624 451.6734619 485.3283691
0.19083333 201.6110229 254.4679413 316.6767578 349.6478882 422.5226746 448.0487366 483.6942749
0.19250001 202.5348053 255.4064026 322.4137268 353.6104431 425.4262695 449.719574 484.5869751
0.194166675 201.3292542 257.4733582 320.9254456 355.0557861 425.6204834 449.7828979 483.2945251
0.19583334 201.5925446 256.4323425 324.4494019 350.1928406 425.3080444 449.5937805 484.5419006
0.197500005 201.4810333 255.4951324 326.051239 354.5004578 424.0201721 451.6187439 483.0815735
0.19916667 201.2186279 255.7566223 327.5127563 352.7685547 424.4910889 449.7717285 480.5174561
0.200833336 200.7128906 254.24263 330.4981079 350.1116638 420.3827515 451.2272034 482.4416199
0.202500016 200.1775513 254.226059 331.095459 351.3789368 424.0718994 449.4387512 484.2839966
0.204166666 200.5095825 254.0241241 333.0923767 352.0473328 422.2650757 448.2684021 481.5639038
0.205833346 201.8906555 254.1396027 332.0444641 352.4859924 425.8720398 450.085144 481.8773193
0.207499996 200.5893555 253.4397278 335.5488892 352.3067627 421.6978149 449.2029419 484.4074097
0.209166676 201.8211212 255.2892303 334.3772278 351.9978943 421.3349609 450.5758057 485.0392456
0.210833341 200.4090729 255.3258514 338.3756409 354.1398926 422.8356323 452.3662109 483.9054565
0.212500006 201.0654144 254.5606842 337.8710938 352.0805359 419.5098267 451.5551758 482.5337524
0.214166671 201.8339844 254.7165222 341.0564575 352.5375366 419.6690674 450.8305054 482.399231
0.215833336 200.0441132 253.7798004 342.5037537 352.5899353 420.3640137 450.1879883 484.9579163
0.217500001 200.8693085 256.1480103 342.3279114 353.4829712 418.0842896 447.1278992 482.9432373
0.219166681 201.3688812 254.0891418 344.2977295 353.0597229 420.0727539 449.6513367 482.9015503
0.220833331 201.2899475 254.4346771 344.5378113 353.3995667 418.8450012 447.0712891 483.1174011
0.222500011 200.663269 254.2605133 346.5697021 355.5457153 419.3755188 445.1726685 481.8770752
0.224166662 202.6383362 254.1178131 344.5266724 353.3104248 418.2282715 448.0934448 483.3558044
0.225833342 202.4537659 252.9151154 345.0093079 354.0796509 422.1054688 447.8424072 483.1300049
0.227500007 203.0107422 254.0090332 342.9070129 356.1683655 420.2193604 449.8345032 484.586731
0.229166672 205.2194519 254.0701599 343.427887 354.3325195 422.0950928 448.6929626 483.1116943
0.230833337 203.321701 254.1599274 342.3199768 353.5942078 421.0609741 446.4045105 484.7137451
0.232500002 200.9533081 255.356308 340.5515442 354.8544312 419.6222229 449.8253174 484.9720459
0.234166667 201.6164856 254.8274689 336.8833923 352.9570923 419.8236389 448.914856 484.1973572
0.235833347 201.8555298 254.1129608 339.3779602 352.3371887 420.1670227 449.2016602 480.7529907
0.237499997 201.3565063 252.8908844 338.7893982 352.8466492 418.7839966 449.6196899 483.6330872
0.239166677 202.9432373 253.7874908 335.5918884 354.0369568 417.5845337 446.707489 482.31073
0.240833327 203.7595673 252.3792419 333.5476074 353.6220703 420.4822388 446.4476929 483.4996033
0.242500007 200.4059906 253.044281 332.5941467 352.3068237 421.2808838 450.6898499 479.3633118
0.244166672 202.1099091 253.5553589 330.3352051 352.9526062 419.1741333 447.6826172 480.4086609
0.245833337 201.14328 252.9936981 327.9447632 354.5697937 420.2488098 446.2387085 479.6993103
0.247500002 201.6627045 253.790802 326.4492798 352.6429749 421.8884888 448.1844482 480.2624512
0.249166668 201.7852173 252.6636047 324.4846191 353.1908875 420.0808105 450.3544312 478.7590637
0.250833333 203.7855225 253.4106903 321.5100098 352.8811035 420.7402954 447.1965942 478.7068481
0.252499998 203.5384827 252.04245 319.2867432 356.0317383 419.4808044 447.7236938 479.1666565
0.254166663 205.8431396 252.3635254 320.8933411 354.0197754 417.8456116 449.6133423 480.5040894
0.255833328 204.6109467 251.2676392 315.9841003 353.9277344 421.5672302 448.7897339 478.5762939
0.257499993 205.2492218 254.0376434 314.605072 355.3264465 420.294342 449.3639526 476.6184998
0.259166658 203.7616425 254.0343475 312.5722656 353.7676697 420.4620056 449.4092102 476.4151611
0.260833353 201.4223175 251.5362854 308.1832275 354.7567749 420.3599854 451.6090698 477.5042114
0.262499988 202.1479187 252.8733063 307.7592468 353.4871521 419.1730042 448.8254089 480.1489258
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0.892499983 199.5571899 279.8506165 300.3286438 353.0187378 431.8797607 459.1383362 485.0316467
0.894166708 200.3410339 278.8574219 301.9847412 352.9476624 430.4321594 455.7405701 486.4910889
0.895833314 199.4186249 278.1616821 300.8112488 352.5196533 429.4458923 457.7912903 485.5774536
0.897500038 200.4022675 278.4454956 300.2853699 354.6852722 429.5334473 457.3562317 485.724823
0.899166703 201.4779968 278.105835 303.3030701 352.6566772 430.2386475 458.0005798 484.8748474
0.900833309 199.5385437 278.7187805 301.2099609 352.0648804 430.1828918 457.2668152 485.8294678
0.902500033 201.3913574 275.9921875 302.1919861 353.3161621 432.5637817 458.9595337 483.5333557
0.904166639 198.5769196 276.517334 302.8081665 353.7614746 427.0639343 457.0531616 480.4575195
0.905833364 199.6069336 276.442688 304.0233154 353.0658569 428.076355 455.4911804 483.3014221
0.907500029 201.3333588 276.5507202 303.0292053 353.1166992 428.1809998 453.788208 484.3627319
0.909166634 200.1138916 274.5960388 305.6833801 352.7821655 429.7831421 455.2953796 481.0730591
0.910833359 200.7271729 274.0801392 306.4424133 353.2437134 428.5971985 454.840271 479.6430359
0.912500024 198.9748688 274.9679871 306.3713989 352.9240723 425.8883667 456.5896606 479.8184814
0.914166689 198.5287018 272.7660522 305.7908325 353.0611267 427.7430115 453.6419983 477.7706299
0.915833354 201.2494354 274.3452759 306.7952271 352.0320435 424.849762 454.0765381 475.1386108
0.91749996 201.2441559 273.5826416 307.3954163 356.8412476 428.021759 456.7902832 477.6870728
0.919166684 200.6873932 273.7592468 307.2971497 354.175354 425.3564758 455.6258545 475.0595093
0.920833349 201.0488129 272.4590149 308.5015259 355.5024719 427.3459473 455.659668 476.556427
0.922500014 199.6035461 272.0006714 310.0344543 354.7374878 424.5722351 456.2792969 472.2902222
0.924166679 200.3691864 272.9716187 307.8566895 354.2650757 422.6107788 454.259613 475.3753052
0.925833345 199.5085907 272.3909302 311.6749878 353.3088074 422.7233582 453.966156 475.4231567
0.92750001 200.3434448 270.970459 310.5603333 352.9447327 423.0838928 454.2172852 474.4830322
0.929166675 201.322876 270.5090942 311.1733704 355.5383301 422.9675903 450.5711975 476.5230103
0.930833399 200.4456482 268.5041809 311.5938721 355.1631165 424.7527161 453.9320679 474.9499512
0.932500005 200.8347473 268.8667603 313.0572205 355.9605713 419.7436218 452.7793274 476.071106
0.93416667 201.8842926 269.723938 314.3722534 354.6187744 421.6861572 451.4974976 478.4391479
0.935833335 200.1377716 269.7956238 314.6351624 353.9047241 420.9750061 452.0325012 478.7646179
0.9375 200.2457886 268.1018066 313.4799805 355.5389709 421.8278809 451.3735657 480.0463562
0.939166725 199.0772552 268.3963928 314.2247314 356.4881592 420.2593994 449.8542175 480.3622437
0.94083333 199.7839508 267.7816467 316.6448364 357.105957 422.6985474 448.847168 481.4200134
0.942499995 200.0545807 266.2028503 314.2995911 356.541687 419.3587341 451.3932495 479.5157166
0.94416672 201.5225983 266.15448 318.3632507 360.5055542 421.0227051 450.8912048 482.7432556
0.945833325 200.6993256 268.657074 317.8019714 355.3388977 417.7480774 448.6898193 484.4826965
0.94750005 200.1703796 264.8187866 318.4447937 357.0563965 417.774231 450.9622803 482.6702271
 0.949166656 200.1418915 267.1788635 317.7527771 357.3398438 418.2221069 449.3456726 485.4804688
0.950833321 203.4778748 264.7121582 320.3106689 358.1559448 421.8228149 447.6386108 485.3995361
0.952500045 201.4076233 265.0508423 319.4884033 357.4028625 419.6703796 449.4949036 482.2935181
0.954166651 200.4808655 263.4498596 320.004364 357.259491 419.473999 447.5095825 480.3868713
0.955833376 202.8928223 263.9776917 318.4874573 356.9831848 420.9814148 451.7133789 481.803772
0.957500041 200.6317749 263.3521118 319.545929 357.1178589 421.4474487 449.537262 482.7326355
0.959166646 201.2200165 262.8734131 318.2543945 357.345459 421.3966675 447.8816528 484.5915833
0.960833371 200.1650696 264.901947 320.84375 359.9022217 419.0803833 447.4511719 482.8034973
0.962499976 200.5745544 264.5893555 320.3232727 353.3829651 420.0062256 450.0076904 483.6701355
0.964166701 201.6352997 261.9324951 320.6483154 355.4870605 418.2791138 450.4838257 484.3329773
0.965833366 199.8758545 261.5758362 320.8650208 358.5947876 420.0716858 449.1227112 486.0098572
0.967499971 199.9809418 261.468689 319.4934998 358.4775085 419.6054688 448.9592896 484.5304871
0.969166696 200.0142517 260.8142395 321.5635681 357.619751 420.9002686 448.9752197 484.7355652
0.970833361 200.245575 260.4855957 322.6335144 356.0334167 419.5493469 449.6870728 482.6080322
0.972500026 199.759201 259.5227661 321.5655212 357.1161804 418.3761292 448.2344971 485.2909241
0.974166691 200.0502472 259.4473572 322.9538574 357.6761169 418.5637207 449.2199707 486.0405884
0.975833297 201.4134674 260.3366089 322.0906372 356.4678345 417.3501892 448.745697 486.5766602
0.977500022 201.1407013 259.7077026 322.8656616 357.6861877 418.4535828 447.828064 488.3465576
0.979166687 199.7279968 258.4254761 321.7875061 357.5545654 418.5653687 448.7357483 490.2709656
0.980833352 199.0480347 259.8667603 320.5197754 356.353241 417.9754944 449.9372253 489.3566895
0.982500017 201.294693 256.3606873 322.763916 357.0213318 416.9781494 448.5579834 490.7650452
0.984166682 200.8731079 258.7539063 320.0678711 356.8131104 418.7359314 451.9703064 489.6964722
0.985833347 200.7870636 257.9199829 319.3083801 356.4320679 418.4756165 452.0892639 494.7838745
0.987500012 200.2173157 257.0731812 318.3115845 356.1694946 417.7961121 450.597229 492.0956421
0.989166677 200.7939758 256.4876099 317.0573425 354.8112183 421.4450378 451.7123413 493.399353
0.990833342 201.0921936 256.7297668 318.0234985 357.0657959 415.9610596 449.932251 494.9593201
0.992500007 198.9855499 256.0226746 315.8744812 355.7034607 415.5956726 453.1643372 494.920166
0.994166672 202.2840271 256.4229736 317.6579285 357.9601135 418.5532532 451.1025696 498.0575562
0.995833337 199.9951782 254.7088318 317.8895569 355.2545166 417.9240112 449.4848328 500.848938
0.997500062 199.5804291 257.1266174 317.0713806 353.9283142 419.7987061 449.8856812 501.4852905
0.999166668 200.2794647 256.0391235 315.7476807 353.5892944 418.2087708 453.2431335 503.3496704
1.000833273 200.0726471 255.7463074 314.6003418 354.6072083 417.315155 450.216217 498.283783

2. Mean Intensities of each sample against concentration of denaturing agent (urea)

Concentration of Urea (M) Mean Intensity (a.u.) Standard Deviation

0 200.7000155 1.596752912
1 260.948364 8.782597295
2 302.8309132 10.46610873
3 354.6433914 2.735622283
4 425.3679945 16.44906372
5 454.5962016 6.01131877
6 477.6299103 11.39946512
GRAPHS:

1. Intensity versus time graph for samples containing different concentrations of urea.
2. Mean Intensities of each sample against concentration of denaturing agent (urea)
 3. Mean Intensities of each sample against concentration of denaturing agent (urea) with
error bars.

Discussion of Results:

Urea breaks the hydrogen bonds between the supersecondary motifs in the tertiary structure of
bovine serum albumin. As a result, the secondary structures like -helices and -sheets are
exposed to the aqueous environment the compactness of the protein molecule is
compromised, causing an increase in the macromolecular size, which has direct implications on
scattering.

Biologically, misfolded proteins are extremely significant in the presentation of disorders like
Alzheimers disease (caused by an accumulation of -amyloid plaques in the CSF lining of the
dura mater), Parkinsons disease, Bovine Spongiform Encephalopathy (Mad Cow disease),
scrapie infection (caused by prion proteins), Amyotrophic Lateral Sclerosis (ALS) [also known
as Lou Gehrings Syndrome] and Huntingtons chorea. Denaturation studies of protein
unfolding are clinically useful in measuring in-vivo concentrations and tolerance thresholds of
naturally occurring or dietary denaturing agents, such as curcumin (present in turmeric).

INFERENCE: