RESULTS
The corrected data from the SANS experiment were used to plot
the graphs in Figures 3.16 and 3.17 using equation 3.7
Figure 3.16: Intensity of scattering as a function of scattering vector for gliadin.
The number and types of residues in gliadin and deamidated gliadin are given in
Tables 3.3 and 3.4, and the number and type of atoms in amino acid residues
at pH 7.0 were calculated as shown in Table 3.5.
Table 3.3: Type and number of residues in alpha/beta
gliadin.
| Ala(A) | 4 | Phe(F) | 8 | Lys(K) | 3 | Pro(P) | 36 | Asn(N) | 5 |
| Thr(T) | 1 | Cys(C) | 4 | Gly(G) | 5 | Leu(L) | 20 | Ser(S) | 14 |
| Gln(Q) | 92 | Val(V) | 13 | Asp(D) | 1 | His(H) | 6 | Tyr(Y) | 7 |
| Met(M) | 1 | Arg(R) | 5 | Trp(W) | 1 | Glu(E) | 5 | Ile(I) | 11 |
Table 3.4: Type and number of residues in deamidated gliadin.
| Ala(A) | 4 | Phe(F) | 8 | Lys(K) | 3 | Pro(P) | 36 | Asn(N) | 0 |
| Thr(T) | 1 | Cys(C) | 4 | Gly(G) | 5 | Leu(L) | 20 | Ser(S) | 14 |
| Gln(Q) | 0 | Val(V) | 13 | Asp(D) | 6 | His(H) | 6 | Tyr(Y) | 7 |
| Met(M) | 1 | Arg(R) | 5 | Trp(W) | 1 | Glu(E) | 97 | Ile(I) | 11 |
Figure 3.17: Intensity of scattering as a function of scattering vector for deamidated gliadin.
The volume of a molecule was calculated as follows:(eg. ethyl alcohol)
MW of C2H5OH=12+3+12+2+16+1=46
Therefore, 1 mole of ethyl alcohol weighs 46g.
One mole contains N molecules =6.023 x 1023molecules.
Therefore, the weight of a single molecule of ethyl alcohol
=MW/N=46/6.023 x 1023g/molecule=7.64 x 10-23g/molecule
Density of ethyl alcohol=0.791 g/cm3.
Volume per molecule(ethyl alcohol) = 7.64 x 10-23 g.molecule-1 / 0.791 g.cm-3
= 9.66 x 10-23 cm3 molecule-1 = 0.97 x 10-22 cm3 molecule-1
Table 3.5: Type and number of atoms in amino-acid residues.
| RESIDUE |
C |
O |
N |
H |
S |
| Lys(K) | 6 | 1 | 2 | 13 | 0 |
| Arg(R) | 6 | 1 | 4 | 13 | 0 |
| His(H) | 6 | 1 | 3 | 6.5 | 0 |
| Ser(S) | 3 | 2 | 1 | 5 | 0 |
| Thr(T) | 4 | 2 | 1 | 7 | 0 |
| Tyr(Y) | 9 | 2 | 1 | 9 | 0 |
| Cys(C) | 3 | 1 | 1 | 5 | 1 |
| Met(M) | 5 | 1 | 1 | 9 | 1 |
| Trp(W) | 11 | 1 | 2 | 10 | 0 |
| Pro(P) | 5 | 1 | 1 | 7 | 0 |
| Phe(F) | 9 | 1 | 1 | 9 | 0 |
| Leu(L) | 6 | 1 | 1 | 11 | 0 |
| Ile(I) | 6 | 1 | 1 | 11 | 0 |
| Val(V) | 5 | 1 | 1 | 9 | 0 |
| Ala(A) | 3 | 1 | 1 | 5 | 0 |
| Asn(N) | 4 | 2 | 2 | 6 | 0 |
| Gln(Q) | 5 | 2 | 2 | 8 | 0 |
| Asp(D) | 4 | 3 | 1 | 4 | 0 |
| Glu(E) | 5 | 3 | 1 | 6 | 0 |
| Gly(G) | 2 | 1 | 1 | 3 | 0 |
The scattering lengths of atoms of the solvent and proteins used in the experiment
are given in Table 3.6.
Table 3.6: Scattering lengths of some atoms(Koester et al., 1991)
| bH | -0.374 x 10-12cm | bC | 0.665 x 10-12cm | bN | 0.94 x 10-12cm |
| bO | 0.58 x 10-12cm | bS | 0.2804 x 10-12cm | bD | 0.6674 x 10-12cm |
The data from Tables 3.5 and 3.6 were used to calculate the scattering length
densities of amino acid residues and the solvent (Table 3.7 and 3.8)(Baldwin, 1988).
Table 3.7: Scattering lengths and densities of amino acid residues
| RESIDUE |
Volume
[Å3] |
Volume
[10-22cm3] |
[10-12cm] |
[10-12cm] |
[1010cm-2] |
[1010cm2] |
| K | 176.2 | 1.76 | 1.586 | 5.752 | 0.900 | 3.268 |
| A | 92.0 | 0.92 | 1.645 | 2.686 | 1.788 | 2.920 |
| F | 203.0 | 2.03 | 4.139 | 5.180 | 2.040 | 2.552 |
| R | 180.8 | 1.81 | 3.466 | 9.714 | 1.915 | 5.367 |
| H | 167.0 | 1.67 | 4.959 | 6.521 | 2.969 | 3.905 |
| S | 99.0 | 0.99 | 2.225 | 4.308 | 2.247 | 4.352 |
| T | 122.0 | 1.22 | 2.142 | 4.224 | 1.760 | 3.462 |
| Y | 204.0 | 2.04 | 4.719 | 6.802 | 2.310 | 3.334 |
| C | 106.0 | 1.06 | 1.930 | 14.013 | 1.820 | 3.786 |
| M | 171.0 | 1.71 | 1.764 | 2.805 | 1.030 | 1.640 |
| W | 238.0 | 2.38 | 6.035 | 8.118 | 2.536 | 3.411 |
| P | 129.0 | 1.29 | 2.227 | 2.227 | 1.726 | 1.726 |
| L | 168.0 | 1.68 | 1.396 | 2.437 | 0.830 | 1.451 |
| I | 169.0 | 1.69 | 1.396 | 2.437 | 0.826 | 1.442 |
| V | 142.0 | 1.42 | 1.479 | 2.520 | 1.040 | 1.775 |
| N | 135.0 | 1.35 | 3.456 | 6.580 | 2.560 | 4.874 |
| Q | 161.0 | 1.61 | 3.373 | 6.497 | 2.095 | 4.035 |
| D | 113.6 | 1.14 | 3.845 | 4.886 | 3.385 | 4.286 |
| E | 140.6 | 1.41 | 3.762 | 4.803 | 2.676 | 3.406 |
| G | 66.0 | 0.66 | 1.728 | 2.769 | 2.618 | 4.195 |
Table 3.8: Scattering lengths and densities of solvent molecules
| Molecule |
Volume
[Å3] |
Volume
[10-22cm3] |
[10-12cm] |
[10-12cm] |
[1010cm-2] |
[1010cm2] |
| H2O | 30.0 | 0.30 | -0.1677 | - | -0.560 | - |
| D2O | 30.0 | 0.30 | - | 1.9153 | - | 6.384 |
| C2H5OH | 96.0 | 0.97 | -0.334 | - | -0.348 | - |
The large difference in neutron scattering between hydrogen and deuterium was
used to contrast match the solvent and the protein molecules using
equation 3.9 (Stuhrmann, 1974). The results are shown
in Tables 3.9 and 3.10 and Figures 3.18 and 3.19.
Table 3.9: Calculations for gliadin samples(G1-G4) containing varying amounts of H2O:D2O
| Molecules |
G1 |
G2 |
G3 |
G4 |
| D2O(%) | 0 | 10 | 20 | 30 |
| H2O(%) | 30 | 20 | 10 | 0 |
| C2H5OH(%) | 70 | 70 | 70 | 70 |
| gliadin conc.(%) | 2.87 | 3.67 | 3.23 | 3.28 |
scattering length density of solvent x 1010cm-2 | -0.4116 | 0.2828 | 0.9772 | 1.6716 |
scattering length density of gliadin x 1010cm-2 | 1.86 | 1.991 | 2.122 | 2.253 |
 | 2.27 | 1.71 | 1.14 | 0.581 |
scattering length density of gliadin at 100% D2O=3.17 x 1010cm-2
Figure 3.18: Mean scattering length density of gliadin as a function of the
scattering length density of H2O/D2O.
Table 3.10: Calculations for deamidated gliadin samples(S1-S6) containing varying amounts of H2O:D2O
| Molecules |
S1 |
S2 |
S3 |
S4 |
S5 |
S6 |
| D2O(%) | 0 | 10 | 20 | 30 | 40 | 50 |
| H2O(%) | 50 | 40 | 30 | 20 | 10 | 0 |
| C2H5OH(%) | 50 | 50 | 50 | 50 | 50 | 50 |
| de.gliadin conc.(%) | 3.68 | 3.61 | 3.67 | 3.47 | 3.76 | 3.71 |
| scattering length density of solventx 1010cm-2 | -0.454 | 0.24 | 0.93 | 1.63 | 2.32 | 3.018 |
| scattering length density of de.gliadinx 1010cm-2 | 2.1 | 2.18 | 2.26 | 2.34 | 2.42 | 2.50 |
| 2.549 | 1.94 | 1.33 | 0.71 | 0.10 | -0.52 |
scattering length density of deamidated gliadin at 100% D2O=2.9 x 1010cm-2
Figure 3.19: Mean scattering length density of deamidated gliadin as a
function of the scattering length density of H2O:D2O.
In calculating the scattering length density of the proteins, it was assumed
that for the 100% D2O, all the labile protons in the protein
were exchanged for deuterium. When proteins are in their native state, it is
only the labile protons exposed to the solvent that are exchanged with deuterium.
The labile protons in the hydrophobic core are not exchanged. Taking this fact
into consideration, a new scattering length density was calculated based on
computer prediction of the most likely labile protons to be exposed to the solvent.
scattering length density of gliadin at 100% D2O=2.83 x 1010cm-2
scattering length density of deamidated gliadin at 100% D2O=2.67 x 1010cm-2
A summary of the calculated results for both gliadin and deamidated gliadin
is shown in Tables 3.11 and 3.12.
Table 3.11: GLIADIN
| residue(r) |
 |
 |
 |
 |
 |
 |
 |
 |
| A | 4 | 2 | 2 | 7.15 | 3.58 | 5.84 | 9.42 | 11.68 |
| F | 8 | - | 8 | 16.32 | - | 20.42 | 20.42 | 20.42 |
| K | 3 | 1 | 2 | 2.7 | 0.9 | 6.54 | 7.44 | 9.804 |
| P | 36 | 2 | 34 | 62.14 | 3.45 | 58.68 | 62.13 | 62.14 |
| T | 1 | 1 | - | 1.76 | 1.76 | - | 1.76 | 3.46 |
| C | 4 | 4 | - | 7.28 | 7.28 | - | 7.28 | 15.14 |
| G | 5 | 1 | 4 | 13.09 | 2.62 | 16.78 | 19.4 | 20.98 |
| L | 20 | 9 | 11 | 16.6 | 7.47 | 15.96 | 23.43 | 29.02 |
| Q | 92 | 17 | 75 | 192.74 | 35.62 | 302.63 | 338.25 | 371.22 |
| V | 13 | 6 | 7 | 13.52 | 6.24 | 12.43 | 18.67 | 23.08 |
| D | 1 | 1 | - | 3.39 | 3.39 | - | 3.39 | 4.29 |
| H | 6 | 6 | - | 17.81 | 17.81 | - | 17.81 | 23.43 |
| M | 1 | 1 | - | 1.03 | 1.03 | - | 1.03 | 1.64 |
| R | 5 | - | 5 | 9.575 | - | 26.84 | 26.84 | 26.84 |
| W | 1 | - | 1 | 2.54 | - | 3.41 | 3.41 | 3.41 |
| E | 5 | 2 | 3 | 13.38 | 5.35 | 10.22 | 15.57 | 17.03 |
| I | 11 | 8 | 3 | 9.09 | 6.61 | 4.33 | 10.94 | 15.86 |
| N | 5 | 2 | 3 | 12.8 | 5.12 | 14.62 | 19.74 | 24.37 |
| S | 14 | 3 | 11 | 31.46 | 6.74 | 47.87 | 54.61 | 60.93 |
| Y | 7 | 1 | 6 | 16.17 | 2.31 | 20.00 | 22.31 | 23.34 |
| Total | 242 | 67 | 175 | 450.53 | 117.27 | 566.55 | 683.85 | 768.06 |
 | | | | 1.86 | | | 2.83 | 3.17 |
Table 3.11: DEAMIDATED GLIADIN
| residue(r) |
|
|
|
|
|
|
|
|
| A | 4 | 2 | 2 | 7.15 | 3.58 | 5.84 | 9.42 | 11.68 |
| F | 8 | - | 8 | 16.32 | - | 20.42 | 20.42 | 20.42 |
| K | 3 | 1 | 2 | 2.7 | 0.9 | 6.54 | 7.44 | 9.804 |
| P | 36 | 2 | 34 | 62.14 | 3.45 | 58.68 | 62.13 | 62.14 |
| T | 1 | 1 | - | 1.76 | 1.76 | - | 1.76 | 3.46 |
| C | 4 | 4 | - | 7.28 | 7.28 | - | 7.28 | 15.14 |
| G | 5 | 1 | 4 | 13.09 | 2.62 | 16.78 | 19.4 | 20.98 |
| L | 20 | 9 | 11 | 16.6 | 7.47 | 15.96 | 23.43 | 29.02 |
| Q | 0 | 0 | 0 | - | - | - | - | - |
| V | 13 | 6 | 7 | 13.52 | 6.24 | 12.43 | 18.67 | 23.08 |
| D | 6 | 3 | 3 | 20.31 | 10.16 | 12.86 | 23.02 | 25.72 |
| H | 6 | 6 | - | 17.81 | 17.81 | - | 17.81 | 23.43 |
| M | 1 | 1 | - | 1.03 | 1.03 | - | 1.03 | 1.64 |
| R | 5 | - | 5 | 9.575 | - | 26.84 | 26.84 | 26.84 |
| W | 1 | - | 1 | 2.54 | - | 3.41 | 3.41 | 3.41 |
| E | 97 | 19 | 78 | 259.57 | 50.84 | 265.67 | 316.51 | 330.38 |
| I | 11 | 8 | 3 | 9.09 | 6.61 | 4.33 | 10.94 | 15.86 |
| N | 0 | 0 | 0 | - | - | - | - | - |
| S | 14 | 3 | 11 | 31.46 | 6.74 | 47.87 | 54.61 | 60.93 |
| Y | 7 | 1 | 6 | 16.17 | 2.31 | 20.00 | 22.31 | 23.34 |
| Total | 242 | 67 | 175 | 508.12 | 128.8 | 517.63 | 646.43 | 707.27 |
| | | | 2.1 | | | 2.67 | 2.9 |
Legend
= total number of residue in protein
= number of residue in hydrophobic core of protein
= number of residue exposed to solvent
= scattering length density for total number of residue in protein at 0%D2O
= scattering length density for residue in hydrophobic core of protein
= scattering length density for residue exposed to 100%D2O
= scattering length density of total residue in protein in 100%D2O
= scattering length density of total residue of denatured protein in 100%D2O
A Guinier analysis was carried out on the data in Figures 3.16 and 3.17 using equation 3.8 (Table 3.13).
Table 3.13: Results of Guinier Analysis
| sample |
 |
 |
 |
 |
 |
 |
 |
| G1 | 2.27 | 1.3±0.095 | 0.59±0.03 | 1.183 | 0.838 | 94.3 | 32 |
| G2 | 1.71 | 1.3±0.08 | 0.45±0.02 | 1.041 | 0.606 | 94.3 | 32 |
| G3 | 1.14 | 0.99±0.07 | 0.34±0.02 | 0.974 | 0.601 | 94.3 | 32 |
| G4 | 0.58 | 0.93±0.07 | 0.32±0.02 | 0.921 | 0.542 | 101.9 | 35 |
| S1 | 2.55 | 2.0±0.25 | 0.408±0.2 | 1.400 | 0.630 | 138.7 | 31.2 |
| S2 | 1.94 | 1.6±0.2 | 0.297±0.1 | 1.260 | 0.540 | 138.7 | 31.2 |
| S3 | 1.33 | 0.81±0.13 | 0.177±0.1 | 0.891 | 0.417 | 138.7 | 31.2 |
| S4 | 0.71 | 0.69±0.09 | 0.049±0.1 | 0.850 | 0.225 | 139.4 | 31.3 |
| S6 | -0.52 | 1.38±0.2 | 0.036±0.1 | 1.160 | 0.187 | 139.4 | 31.3 |
