Progetto: LIFE10 ENV/IT/000364

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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
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• ACTION 1
• FAT AGENTS
• Characterization of fat agents (solfochloroparaffi
  n, SCP Chloroparaffins CP30, CP44)
• Study of the interaction of fat agents with skin
  proteins (collagen, gelatin, skin powder)

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FTIR spectra of CP, CP30 (C18) CP44 (C14-C17)
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Comparison between FTIR spectra of paraffin
(C18-C20), CP30 and CP44
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Study of the inteaction of fat agents with skin
proteins (collagen, gelatin, skin
powder) Experimental plan
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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 isopropanol
In progress!
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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.
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
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
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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.
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METHODS
Infrared spectroscopy (ATR-FTIR) Computational
study Thermogravimetric Analysis (TGA)
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Is gelatin a good model? FTIR spectra of
gelatins and skin powder
Amide II
Amide I
Gelatin B
Gelatin Merck
Skin powder
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FTIR spectra of dried skin powder spiked with
10-20-30 SCP
SP 30 SCP
SP 20 SCP
SP 10 SCP
SP
SCP
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FTIR spectra of dried skin powder spiked with
10-20-30 SCP after esane extraction
SP 20 SCP
SP 30 SCP
SP 10 SCP
SP
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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 11 MilliQ (SCP/CP 50 mg/mL) (emulsion)
11 MeOH/MilliQ emulsion
Gelatin A and B (Sigma) (100 mg 1 mL emulsion)
blank
2h30 incubation time RT and stirring
pH monitoring and adjustment
Esane extraction
Esane extraction
drying
drying
FTIR analysis TGA analysis
pH changed and was adjusted only in the case of
Gelatin A treated with SCP in isopropanol/H2O
emulsion
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FTIR spectra of Gelatin B 50 CP30 (C18) /CP44
(C14-C17) MeOH/water emulsion after esane
extraction and drying
CP30
CP44
blank
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FTIR spectra of Gelatin B 50 SCP in MeOH/water
emulsion after esane extraction and drying
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SCP
blank
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Semi-quantitative spectroscopic parameters to
evaluate SCP/CP-protein interactions summary of
gelatin and skin powder reactivity
-SO2- SO stretching vibration typical of SCP
Micelle size??
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Semi-quantitative spectroscopic parameters to
evaluate SCP/CP-protein interactions summary of
gelatin and skin powder reactivity
C-Cl stretching vibrations typical of CP
CP44
CP30
A B SP
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FTIR spectra of dried skin powder 50 CP30 in
MeOH or isopropanol after esane extraction and
drying
1635
524
1236
1030
1447
1538
1334
1203
1080
3283
2931
1400
1163
972
3070
SP
2926
609
2855
1459
658
727
1378
1263
898
1123
1745
524
CP30
A
1631
Amine C-N stretch ??? (1360-1020) Ether C-O
stretch??
1449
1235
1544
1022
1397
3287
1336
1204
2928
1080
3075
SPCP30 in MeOH
524
1631
1235
1448
1538
1334
1080
2927
3284
1399
1161
1030
2857
3072
SPCP30 in Isopr
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
450,0
cm-1
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Collagen model for computational study
Collagen microfibril segment (CMS)
ACE GLY PRO MET GLY PRO SER GLY PRO ARG GLY LEU
HPR GLY PRO HPR GLY ALA HPR GLY PRO GLN GLY PHE
NME ACE GLY PRO MET GLY LEU MET GLY PRO ARG GLY
PRO HPR GLY ALA SER GLY ALA HPR GLY PRO GLN GLY
PHE NME ACE GLY PRO MET GLY PRO SER GLY PRO ARG
GLY LEU HPR GLY PRO HPR GLY ALA HPR GLY PRO GLN
GLY PHE NME ACE GLY ALA ARG GLY PRO SER GLY PRO
GLN GLY PRO SER GLY PRO HPR GLY PRO LYS GLY ASN
SER GLY GLU NME ACE GLY PRO ARG GLY ILE HPR GLY
PRO VAL GLY ALA ALA GLY ALA THR GLY ALA ARG GLY
LEU VAL GLY GLU NME ACE GLY ALA ARG GLY PRO SER
GLY PRO GLN GLY PRO SER GLY PRO HPR GLY PRO LYS
GLY ASN SER GLY GLU NME ACE GLY LEU GLN GLY PRO
HPR GLY PRO HPR GLY SER HPR GLY GLU GLN GLY PRO
SER GLY ALA SER GLY PRO NME ACE GLY LEU GLN GLY
LEU HPR GLY LEU ALA GLY HID HID GLY ASP GLN GLY
ALA HPR GLY ALA VAL GLY PRO NME ACE GLY LEU GLN
GLY PRO HPR GLY PRO HPR GLY SER HPR GLY GLU GLN
GLY PRO SER GLY ALA SER GLY PRO NME ACE GLY ARG
VAL GLY PRO HPR GLY PRO SER GLY ASN ALA GLY PRO
HPR GLY PRO HPR GLY PRO ALA GLY LYS NME ACE GLY
ARG THR GLY THR HPR GLY PRO SER GLY ILE SER GLY
PRO HPR GLY PRO HPR GLY PRO ALA GLY LYS NME ACE
GLY ARG VAL GLY PRO HPR GLY PRO SER GLY ASN ALA
GLY PRO HPR GLY PRO HPR GLY PRO ALA GLY LYS
NME ACE GLY PRO ALA GLY PRO HPR GLY GLU ALA GLY
LYS HPR GLY GLU GLN GLY VAL HPR GLY ASP LEU GLY
ALA NME ACE GLY PRO ALA GLY THR ALA GLY GLU ALA
GLY LYS HPR GLY GLU ARG GLY ILE HPR GLY GLU PHE
GLY LEU NME ACE 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
pH 6-7 CMS net charge 4 Collagen dimer Agent
SCP C20 (22 molecules) with 3 SC groups 14981
water molecules 8 Cl- T37C
Sulpho Chlorinated Paraffin SCP_C20. Molecular
Electrostatic Potential (MEP) on the solvent
accessible surface (red areas negative
potential, blue areas positive potential).
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Collagen computational study preliminary results
Evolution of the Root Mean Square Deviation of
the trace (Ca) 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
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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.
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Future actions (ACTIONS 3-4)
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Treatment Gelatin 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!
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In summary

• 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

N benzyl methyl amine
1,2,3,4-tetrahydroisoquinoline
N-ethyl benzyl amine
NHR2 SCP ? SCP-NR2 HCl HPLC/UV/fluorescence
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