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Progetto : LIFE10 ENV/IT/000364 «ECOFATTING» « Environmentally friendly natural products instead of cloroparaffines in the fatting phase of the tanning cycle » EMILIA BRAMANTI ICCOM-PISA, CNR. 6 month Meeting – CNR, ICCOM PISA premises, June 22th 2012. ACTION 1 FAT AGENTS - PowerPoint PPT Presentation
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Progetto: LIFE10 ENV/IT/000364 «ECOFATTING»
«Environmentally friendly natural products instead of cloroparaffines in the fatting
phase of the tanning cycle»
EMILIA BRAMANTIICCOM-PISA, CNR
6 month Meeting – CNR, ICCOM PISA premises, June 22th 2012
ACTION 1FAT AGENTS
•Characterization of fat agents (solfochloroparaffin, SCP; Chloroparaffins CP30, CP44)
•Study of the interaction of fat agents with skin proteins (collagen, gelatin, skin powder)
3
FTIR spectra of CP, CP30 (C18) CP44 (C14-C17)
1800 1600 1400 1200 1000 800 600 500cm-1
SCP
CP30
CP44
1740
1446
1379
1262
1123
903
791
740
652608
1745
1459
13781263
1123898
727
658
609
1741
1461
13651158
691
621
586564521
3000 2900 2800 2700cm-1
A
SCP
CP30
CP44
2933
2863
2926
28552924
2855
-SO2- S=O stretchingabsorptions
C-Cl absorptions
Comparison between FTIR spectra of paraffin (C18-C20), CP30 and CP44
1500 1400 1300 1200 1100 1000 900 800 700 600 500cm-1
A
Paraffin
CP44
CP30
1459
1378
1263
1123
898
727
658
609
1446
1379
1262
1123
903791
740
652
608
1466
1377
1120889
721
y = 0.0123x2 + 0.0855x - 2E-13R² = 1
y = 0.0223x2 + 0.2707x - 3E-13R² = 1
0
10
20
30
40
50
60
0 20 40 60
pea
k ar
ea
% Cl
3040 2960 2800
A
P
CP44
CP30
2926
2855
29332957
2916
2849
652-607 peak
1261 peak
Study of the inteaction of fat agents with skin proteins (collagen, gelatin, skin powder):
Experimental plan
Treatment Gelatin
Merk
Gelatin Sigma B
(bovine skin)
Gelatin Sigma A
(porcine skin)
Skin
powder
Collagen
Spike on dried powders with
SCP 10-20-30%
Spike on wet powders with
SCP 10-20-30%
Spike on dried powders with
SCP 10-20-30%+ esane
extraction+ drying
Dried powders+
SCP/CP30/CP44 in
MeOH/H2O or
isopropanol/H2O emulsion
Dried Powders+
SCP/CP30/CP44 in MeOH or
isopropanol
Wet powders (40% water)+
SCP/CP30/CP44 in MeOH or
isopropanolIn progress!
Gelatin is a heterogeneous mixture of water-soluble proteins of high average molecular masses, present in collagen. The proteins are extracted by boiling skin, tendons, ligaments, bones, etc. in water. In skin fibrous proteins are 96.5% and among these collagen represents 98% (1% elastin, 1% keratin). Type A gelatin is derived from porcine acid-cured tissue Type B gelatin is derived from bovine lime-cured tissue.
The charge on a gelatin molecule and its isoelectric point are primarily due to the carboxyl, amino, and guanidino groups on the side chains
* Bloom number is an indication of the strength of a gel formed from a solution of known concentration. It is correlated with MW
Sample Free carboxyl
groups
Isoelectric
point (pI)
1.5%
solution pH
Bloom
number*
Average
MW (Da)
Gelatin A from porcine
skin (G-2500)
78-80
millimol/100 g
protein
7.0-9.0 3.8-5.5 300 50.000-
100.000
Gelatin B from bovine
skin (G-9382)
100-115 millimol / 100 g protein
4.7-5.2 5.0-7.5 225 40.000-
50.000
Collagen
67 nm
The helical conformation of each chain depends on the presence of glycine (GLY) every two residues and on the high content of proline (PRO) and hydroxyproline (HPR). HPR increases significantly the conformational stability of a collagen triple helix.
In all fibrillar collagens the chains include a continuos sequence of about 300 GLY-X-Y triplets flanked by terminal globular domains (telopeptides). Depending on the collagen type and on the tissue, triple helices can be homo- or heterotrimers.
Telopeptides are of particular importance in the formation of collagen fibrils. These segments do not assume the triple-helical confromation and contain the unusual amino acidssuch as hydroxylysine. Covalent cross-links between adjacent molecules stabilize the side-by-side packing of collagen molecules and generate strong fibrils.
Polar and hydrophobic residues are periodically clustered along the sequence of collagen I (e.g.) every 234 residues.
Collagens are one of the most crucial components of the extracellular matrix
Water is one of the major contributors to the enthalpy of denaturation of collagen and the enhanced collagen chemical and hydrothermal stability in the presence of modifying agent molecules is a result of fiber cross-linking.
Polypeptide chain
Triple stranded collagen
1.5 nm
330 nm
Collagen fibril
10-300 nm
Collagen Microfibril Segment
METHODS
Infrared spectroscopy (ATR-FTIR)
Computational study
Thermogravimetric Analysis (TGA)
Is gelatin a good model?
FTIR spectra of gelatins and skin powder
1800 1700 1600 1500 1400 1300 1200 1100 1000 900cm-1
A
1635
1538
14471400
1334
1236
10801030
972
16291523
14421398
1330
1237
1161
10791028
Gelatin MerckGelatin B
Skin powder
Amide I
Amide II
FTIR spectra of dried skin powder spiked with 10-20-30% SCP
3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600cm-1
A
1463
1364
1157
3285
3073
2924
2854
1633
15401457
1237
1159
10801031
851
690
586564 529
1363
y = 0.0075x2 + 0.065x + 1448.2R2 = 0.9899
1447
1448
1449
1450
1451
1452
1453
1454
1455
1456
1457
1458
0 10 20 30 40
cm-1
SCP% added
shift band CH2 bending
SCP
SP+ 10% SCP
SP+ 20% SCP
SP+ 30% SCP
SP
FTIR spectra of dried skin powder spiked with 10-20-30% SCP after esane extraction
3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600cm-1
A 3283
3067
2927
2859
1633
1532
14481367
12321159
1080
8522924
2854
1463
1364
1157
691
-5
0
5
10
15
20
25
30
35
0 10 20 30
pe
ak
are
a
SCP %
CH2 stretching area1448 area1367 area1159 area
SP
SP+ 10% SCP
SP+ 20% SCP
SP+ 30% SCP
13
New protocol to improve sample homogeneity: solubilization of SCP and CP in MeOH or isopropanol and treatment of gelatins with the MeOH/H2O or isopropanol/H2O emulsion SCP/CP
500 mg in 5 mL MeOH(100 mg/mL)
Diulted 1:1 MilliQ(SCP/CP 50 mg/mL)(emulsion)
Gelatin A and B (Sigma)(100 mg + 1 mL emulsion)
1:1 MeOH/MilliQemulsion
Esane extraction
Esane extraction
drying
dryingFTIR analysisTGA analysis
blankpH monitoring and adjustment*
*pH changed and was adjusted only in the case of Gelatin A treated with SCP in isopropanol/H2O emulsion
2h30’ incubation time RT and stirring
1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500cm-1
A
1633
1541
1458
1378
1310
1237 (1263 cm-1 in pure CP)
1081
1033
971 899
726
657
6101629
1547
1453
1378
13381239
1203
1160
1034
936
591563529
1742
FTIR spectra of Gelatin B + 50% CP30 (C18) /CP44 (C14-C17) MeOH/water emulsion after esane extraction and drying
CP30
CP44
blank
3600 3200 2800
A
2925
2856
2926
2860
3292
3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600cm-1
A
3297
3076
2925
2855 1631
1545
1459
1235
1159
691
621
586
564530
2934
2864
1740
1080
1031
15
SCP
FTIR spectra of Gelatin B + 50% SCP in MeOH/water emulsion after esane extraction and drying
blank
1364
GA+SCP/MeOH/H2O
GA+SCP/isopropanol/H2O*
GB+SCP/MeOH/H2O
GB+SCP/isopropanol/H2O
SP+SCP/MeOH/H2O
SP+SCP/isopropanol/H2O
0.0
0.5
1.0
1.5
2.0 OD1364/OD1630
Semi-quantitative spectroscopic parameters to evaluate SCP/CP-protein interactions: summary of gelatin and skin powder reactivity
-SO2- S=O stretching vibration typical of SCP
Micelle size??
Semi-quantitative spectroscopic parameters to evaluate SCP/CP-protein interactions: summary of gelatin and skin powder reactivity
CH2 stretching vibrations of SCP and CP
GA+SCP/MeOH/H2O
GA+SCP/isopropanol/H2O*
GB+SCP/MeOH/H2O
GB+SCP/isopropanol/H2O
SP+SCP/MeOH/H2O
SP+SCP/isopropanol/H2O --
GA+CP30/MeOH/H2O
GA+CP30/isopropanol/H2O*
GB+CP30/MeOH/H2O
GB+CP30/isopropanol/H2O
SP+CP30/MeOH/H2O
SP+CP30/isopropanol/H2O --
GA+CP44/MeOH/H2O
GA+CP44/isopropanol/H2O*
GB+CP44/MeOH/H2O
GB+CP44/isopropanol/H2O
SP+CP44/MeOH/H2O
SP+CP44/isopropanol/H2O --
GA/MeOH/H2O
GA/isopropanol/H2O
GB/MeOH/H2O
GB/isopropanol/H2O
SP/MeOH/H2O
SP/isopropanol/H2O
0
20
40
60
80
100
A
CH2 stretching peak area
SCPCP30 CP44
blank
A B SP
Semi-quantitative spectroscopic parameters to evaluate SCP/CP-protein interactions: summary of gelatin and skin powder reactivity
CH2 bending vibrations of SCP and CP
GA+SCP/MeOH/H2O
GA+SCP/isopropanol/H2O*
GB+SCP/MeOH/H2O
GB+SCP/isopropanol/H2O
SP+SCP/MeOH/H2O
SP+SCP/isopropanol/H2O --
GA+CP30/MeOH/H2O
GA+CP30/isopropanol/H2O*
GB+CP30/MeOH/H2O
GB+CP30/isopropanol/H2O
SP+CP30/MeOH/H2O
SP+CP30/isopropanol/H2O --
GA+CP44/MeOH/H2O
GA+CP44/isopropanol/H2O*
GB+CP44/MeOH/H2O
GB+CP44/isopropanol/H2O
SP+CP44/MeOH/H2O
SP+CP44/isopropanol/H2O --
GA/MeOH/H2O
GA/isopropanol/H2O
GB/MeOH/H2O
GB/isopropanol/H2O
SP/MeOH/H2O
SP/isopropanol/H2O
0
5
10
15
20
25
30
35
1458 Peak area
SCPCP30 CP44
blank
Semi-quantitative spectroscopic parameters to evaluate SCP/CP-protein interactions: summary of gelatin and skin powder reactivity
C-Cl stretching vibrations typical of CP
--
GA+CP30/MeOH/H2O
GA+CP30/isopropanol/H2O*
GB+CP30/MeOH/H2O
GB+CP30/isopropanol/H2O
SP+CP30/MeOH/H2O
SP+CP30/isopropanol/H2O --
GA+CP44/MeOH/H2O
GA+CP44/isopropanol/H2O*
GB+CP44/MeOH/H2O
GB+CP44/isopropanol/H2O
SP+CP44/MeOH/H2O
SP+CP44/isopropanol/H2O0
5
10
15
20
25
30
35
40
714-540 peak area
CP30 CP44
A B SP
4000,0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450,0cm-1
A
2924
28541463
13641157
691623
587
564
534
520
3283
30702931
163515381447
1400
13341236
12031163
10801030
972
524
32863075
2932
2875
162915421449
1368
1336
1236
1201
1159
1079
1033
984
523
32853073
2933
16301540
14471398
13351235
1204
11611079
1030
972
945
521
SCP
SP
SP+SCP in MeOH
SP+SCP in Isopr
FTIR spectra of dried skin powder + 50% SCP in MeOH or isopropanol after esane extraction and drying
3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600cm-1
A
2933
28631740
1446
1379
1262
11231056
984903
791740 652
608
32833070
2931
16351538 1447
14001334
1236
12031163
1080
1030972
524
3302
3077
2939
2831
16351547
1448
1399
13361237
1205
1163
1021 554528
3284
3069
2933
16341534
1445
1399
1334
12341161
1080
1030
524
CP44
SP
SP+CP44 in MeOH
SP+CP44 in Isopr
FTIR spectra of dried skin powder + 50% CP44 in MeOH or isopropanol after esane extraction and drying
Amine C-N stretch ??? (1360-1020)Ether C-O stretch??
4000,0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450,0cm-1
A
3283
3070
2931
1635
1538 1447
14001334
1236
1203
11631080
1030
972
524
2926
2855
1745
1459
137812631123
898
727658
609
3287
30752928
1631154414491397
1336
1235
12041080
1022
524
32843072
2927
2857
163115381448
1399
13341235
11611080
1030
524
CP30
SP
SP+CP30 in MeOH
SP+CP30 in Isopr
FTIR spectra of dried skin powder + 50% CP30 in MeOH or isopropanol after esane extraction and drying
Amine C-N stretch ??? (1360-1020)Ether C-O stretch??
Collagen model for computational study
pH 6-7CMS net charge +4
Collagen dimer
Agent : SCP C20 (22 molecules) with 3 SC groups
14981 water molecules8 Cl-
T=37°C
ACE GLY PRO MET GLY PRO SER GLY PRO ARG GLY LEU HPR GLY PRO HPR GLY ALA HPR GLY PRO GLN GLY PHE NMEACE GLY PRO MET GLY LEU MET GLY PRO ARG GLY PRO HPR GLY ALA SER GLY ALA HPR GLY PRO GLN GLY PHE NMEACE GLY PRO MET GLY PRO SER GLY PRO ARG GLY LEU HPR GLY PRO HPR GLY ALA HPR GLY PRO GLN GLY PHE NMEACE GLY ALA ARG GLY PRO SER GLY PRO GLN GLY PRO SER GLY PRO HPR GLY PRO LYS GLY ASN SER GLY GLU NMEACE GLY PRO ARG GLY ILE HPR GLY PRO VAL GLY ALA ALA GLY ALA THR GLY ALA ARG GLY LEU VAL GLY GLU NMEACE GLY ALA ARG GLY PRO SER GLY PRO GLN GLY PRO SER GLY PRO HPR GLY PRO LYS GLY ASN SER GLY GLU NMEACE GLY LEU GLN GLY PRO HPR GLY PRO HPR GLY SER HPR GLY GLU GLN GLY PRO SER GLY ALA SER GLY PRO NMEACE GLY LEU GLN GLY LEU HPR GLY LEU ALA GLY HID HID GLY ASP GLN GLY ALA HPR GLY ALA VAL GLY PRO NMEACE GLY LEU GLN GLY PRO HPR GLY PRO HPR GLY SER HPR GLY GLU GLN GLY PRO SER GLY ALA SER GLY PRO NMEACE GLY ARG VAL GLY PRO HPR GLY PRO SER GLY ASN ALA GLY PRO HPR GLY PRO HPR GLY PRO ALA GLY LYS NMEACE GLY ARG THR GLY THR HPR GLY PRO SER GLY ILE SER GLY PRO HPR GLY PRO HPR GLY PRO ALA GLY LYS NMEACE GLY ARG VAL GLY PRO HPR GLY PRO SER GLY ASN ALA GLY PRO HPR GLY PRO HPR GLY PRO ALA GLY LYS NMEACE GLY PRO ALA GLY PRO HPR GLY GLU ALA GLY LYS HPR GLY GLU GLN GLY VAL HPR GLY ASP LEU GLY ALA NMEACE GLY PRO ALA GLY THR ALA GLY GLU ALA GLY LYS HPR GLY GLU ARG GLY ILE HPR GLY GLU PHE GLY LEU NMEACE GLY PRO ALA GLY PRO HPR GLY GLU ALA GLY LYS HPR GLY GLU GLN GLY VAL HPR GLY ASP LEU GLY ALA NME
Amino Acid Content of the CMS
Sulpho Chlorinated Paraffin SCP_C20. Molecular Electrostatic Potential (MEP) on the solvent accessible surface (red areas: negative potential, blue areas: positive potential).
Collagen microfibril segment(CMS)
Collagen computational study: preliminary results
Evolution of the Root Mean Square Deviation of the trace (C) in relation to the starting conformation of the CMS.
The complex is stabilized by the presence of the cross-linking agent and does not change substantially in the last 7.5 ns of the production run
Collagen computational study: preliminary results
CMSs (ribbon or backbone atoms) surrounded by SCP_C20. Spatial distribution contours of SCP oxygen and chlorine atoms
All solvent species are found in the first solvation shell around the bundles where they are in direct contact with the protein residues.
The bundles inflate is due to water activity but the fibril conformation is maintained due to the presence of the cross-linking agents.
Water molecules surround the bundles but are also found between the helices.
SCP molecules are located prevalently between the CMSs and in the terminus regions of the chains and their oxygen atoms interact with the amine groups of ARG and GLN residues.
Future actions
(ACTIONS 3-4)
TreatmentGelatin
Merk
Gelatin Sigma B
(bovine skin)
Gelatin Sigma
A (porcine
skin)
Skin
powder
Collagen Leather
samples
(COLORTEX ,
INESCOP)
Spike on dried powders with
SCP 10-20-30%
….
Spike on wet powders with SCP
10-20-30%
….
Spike on dried powders with
SCP 10-20-30%+ esane
extraction+ drying
……
Dried powders+ SCP/CP30/CP44
in MeOH/H2O or
isopropanol/H2O emulsion
…………
Dried Powders+ SCP/CP30/CP44
in MeOH or isopropanol
…………..
Wet powders (40% water)+
SCP/CP30/CP44 in MeOH or
isopropanol
………….
Commercial products/ Natural
products (SERICHIM)/different
conditions and %
(COLORTEX , INESCOP)
…. …. ….. …. …….. ……………
In progress!
•Conformational analysis of collagen•Modelling and computational study of the interaction of SCP and CP with collagen•Characterization of natural products developed by SERICHIM•Study of the interaction of natural products with Gelatin A, gelatin B, skin powder, collagen, leather samples from COLORTEX/INESCOP.•Development of analytical methods for titration of -SO2Cl groups in SCP and natural products
1,2,3,4-tetrahydroisoquinolineN-ethyl benzyl amine
NHR2 + SCP SCP-NR2 +HCl HPLC/UV/fluorescence
In summary:
N benzyl methyl amine
ECOFATTING PEOPLE @ ICCOM-PI
Emilia BramantiAlessandro D’Ulivo
Massimo Onor
Manuela CempiniValentina Della PortaSusanna Monti
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