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)4Phe(F)8Lys(K)3Pro(P)36Asn(N)5
Thr(T)1Cys(C)4Gly(G)5Leu(L)20Ser(S)14
Gln(Q)92Val(V)13Asp(D)1His(H)6Tyr(Y)7
Met(M)1Arg(R)5Trp(W)1Glu(E)5Ile(I)11

Table 3.4: Type and number of residues in deamidated gliadin.
Ala(A)4Phe(F)8Lys(K)3Pro(P)36Asn(N)0
Thr(T)1Cys(C)4Gly(G)5Leu(L)20Ser(S)14
Gln(Q)0Val(V)13Asp(D)6His(H)6Tyr(Y)7
Met(M)1Arg(R)5Trp(W)1Glu(E)97Ile(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)612130
Arg(R)614130
His(H)6136.50
Ser(S)32150
Thr(T)42170
Tyr(Y)92190
Cys(C)31151
Met(M)51191
Trp(W)1112100
Pro(P)51170
Phe(F)91190
Leu(L)611110
Ile(I)611110
Val(V)51190
Ala(A)31150
Asn(N)42260
Gln(Q)52280
Asp(D)43140
Glu(E)53160
Gly(G)21130

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 10-12cmbC0.665 x 10-12cmbN0.94 x 10-12cm
bO0.58 x 10-12cmbS0.2804 x 10-12cmbD0.6674 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
3]
Volume
[10-22cm3]

[10-12cm]

[10-12cm]

[1010cm-2]

[1010cm2]
K176.21.761.5865.7520.9003.268
A92.00.921.6452.6861.7882.920
F203.02.034.1395.1802.0402.552
R180.81.813.4669.7141.9155.367
H167.01.674.9596.5212.9693.905
S99.00.992.2254.3082.2474.352
T122.01.222.1424.2241.7603.462
Y204.02.044.7196.8022.3103.334
C106.01.061.93014.0131.8203.786
M171.01.711.7642.8051.0301.640
W238.02.386.0358.1182.5363.411
P129.01.292.2272.2271.7261.726
L168.01.681.3962.4370.8301.451
I169.01.691.3962.4370.8261.442
V142.01.421.4792.5201.0401.775
N135.01.353.4566.5802.5604.874
Q161.01.613.3736.4972.0954.035
D113.61.143.8454.8863.3854.286
E140.61.413.7624.8032.6763.406
G66.00.661.7282.7692.6184.195

Table 3.8: Scattering lengths and densities of solvent molecules
Molecule Volume
3]
Volume
[10-22cm3]

[10-12cm]

[10-12cm]

[1010cm-2]

[1010cm2]
H2O30.00.30-0.1677--0.560-
D2O30.00.30-1.9153-6.384
C2H5OH96.00.97-0.334--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(%)0102030
H2O(%)3020100
C2H5OH(%)70707070
gliadin conc.(%)2.873.673.233.28
scattering length density of solventx 1010cm-2-0.41160.28280.97721.6716
scattering length density of gliadinx 1010cm-21.861.9912.1222.253
2.271.711.140.581
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(%)01020304050
H2O(%)50403020100
C2H5OH(%)505050505050
de.gliadin conc.(%)3.683.613.673.473.763.71
scattering length density of solventx 1010cm-2-0.4540.240.931.632.323.018
scattering length density of de.gliadinx 1010cm-22.12.182.262.342.422.50
2.5491.941.330.710.10-0.52
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)
A4227.153.585.849.4211.68
F8-816.32-20.4220.4220.42
K3122.70.96.547.449.804
P3623462.143.4558.6862.1362.14
T11-1.761.76-1.763.46
C44-7.287.28-7.2815.14
G51413.092.6216.7819.420.98
L2091116.67.4715.9623.4329.02
Q921775192.7435.62302.63338.25371.22
V136713.526.2412.4318.6723.08
D11-3.393.39-3.394.29
H66-17.8117.81-17.8123.43
M11-1.031.03-1.031.64
R5-59.575-26.8426.8426.84
W1-12.54-3.413.413.41
E52313.385.3510.2215.5717.03
I11839.096.614.3310.9415.86
N52312.85.1214.6219.7424.37
S1431131.466.7447.8754.6160.93
Y71616.172.3120.0022.3123.34
Total24267175450.53117.27566.55683.85768.06
1.862.833.17

Table 3.11: DEAMIDATED GLIADIN
residue(r)
A4227.153.585.849.4211.68
F8-816.32-20.4220.4220.42
K3122.70.96.547.449.804
P3623462.143.4558.6862.1362.14
T11-1.761.76-1.763.46
C44-7.287.28-7.2815.14
G51413.092.6216.7819.420.98
L2091116.67.4715.9623.4329.02
Q000-----
V136713.526.2412.4318.6723.08
D63320.3110.1612.8623.0225.72
H66-17.8117.81-17.8123.43
M11-1.031.03-1.031.64
R5-59.575-26.8426.8426.84
W1-12.54-3.413.413.41
E971978259.5750.84265.67316.51330.38
I11839.096.614.3310.9415.86
N000-----
S1431131.466.7447.8754.6160.93
Y71616.172.3120.0022.3123.34
Total24267175508.12128.8517.63646.43707.27
2.12.672.9

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
G12.271.3±0.0950.59±0.031.1830.83894.332
G21.711.3±0.080.45±0.021.0410.60694.332
G31.140.99±0.070.34±0.020.9740.60194.332
G40.580.93±0.070.32±0.020.9210.542101.935
S12.552.0±0.250.408±0.21.4000.630138.731.2
S21.941.6±0.20.297±0.11.2600.540138.731.2
S31.330.81±0.130.177±0.10.8910.417138.731.2
S40.710.69±0.090.049±0.10.8500.225139.431.3
S6-0.521.38±0.20.036±0.11.1600.187139.431.3