1. 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

2. ACTION 1 FAT 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. 3 FTIR spectra of CP, CP30 (C18) CP44 (C14-C17)

4. Comparison between FTIR spectra of paraffin (C18-C20), CP30 and CP44

5. Study of the inteaction of fat agents with skin proteins (collagen, gelatin, skin powder): Experimental plan

6. 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!

7. 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 7.0-9.03.8-5.5300 50.000- 100.000 Gelatin B from bovine skin (G-9382) 100-115 millimol / 100 g protein 4.7-5.25.0-7.522540.000- 50.000

8. 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

9. METHODS Infrared spectroscopy (ATR-FTIR) Computational study Thermogravimetric Analysis (TGA)

10. Is gelatin a good model? FTIR spectra of gelatins and skin powder Gelatin Merck Gelatin B Skin powder Amide I Amide II

11. FTIR spectra of dried skin powder spiked with 10-20-30% SCP SCP SP+ 10% SCP SP+ 20% SCP SP+ 30% SCP SP

12. FTIR spectra of dried skin powder spiked with 10-20-30% SCP after esane extraction 3600320028002400200018001600140012001000800600 cm-1 A 3283 3067 2927 2859 1633 1532 1448 1367 1232 1159 1080 852 2924 2854 1463 1364 1157 691 SP SP+ 10% SCP SP+ 20% SCP SP+ 30% SCP

13. 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/MilliQ emulsion Esane extraction drying FTIR analysis TGA analysis blank pH 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

14. FTIR spectra of Gelatin B + 50% CP30 (C18) /CP44 (C14-C17) MeOH/water emulsion after esane extraction and drying CP30 CP44 blank 360032002800 A 2925 2856 2926 2860 3292

15. 3600320028002400200018001600140012001000800600 cm-1 A 3297 3076 2925 2855 1631 1545 1459 1235 1159 691 621 586 564 530 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

16. Semi-quantitative spectroscopic parameters to evaluate SCP/CP- protein interactions: summary of gelatin and skin powder reactivity -SO 2 - S=O stretching vibration typical of SCP Micelle size??

17. Semi-quantitative spectroscopic parameters to evaluate SCP/CP- protein interactions: summary of gelatin and skin powder reactivity CH 2 stretching vibrations of SCP and CP SCP CP30 CP44 blank A B SP

18. Semi-quantitative spectroscopic parameters to evaluate SCP/CP- protein interactions: summary of gelatin and skin powder reactivity CH 2 bending vibrations of SCP and CP SCP CP30 CP44 blank

19. Semi-quantitative spectroscopic parameters to evaluate SCP/CP- protein interactions: summary of gelatin and skin powder reactivity C-Cl stretching vibrations typical of CP CP30 CP44 A B SP

22. 4000,03600320028002400200018001600140012001000800600450,0 cm-1 A 3283 3070 2931 1635 1538 1447 1400 1334 1236 1203 1163 1080 1030 972 524 2926 2855 1745 1459 13781263 1123 898 727 658 609 3287 3075 2928 1631 1544 1449 1397 1336 1235 1204 1080 1022 524 3284 3072 2927 2857 1631 1538 1448 1399 1334 1235 1161 1080 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??

23. Collagen model for computational study pH 6-7 CMS net charge +4 Collagen dimer Agent : SCP C20 (22 molecules) with 3 SC groups 14981 water molecules 8 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 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 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)

24. 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

25. 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.

26. Future actions (ACTIONS 3-4)

27. 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!

28. 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 -SO 2 Cl groups in SCP and natural products 1,2,3,4-tetrahydroisoquinoline N-ethyl benzyl amine NHR 2 + SCP  SCP-NR 2 +HCl HPLC/UV/fluorescence In summary: N benzyl methyl amine

29. ECOFATTING PEOPLE @ ICCOM-PI Emilia Bramanti Alessandro D’Ulivo Massimo Onor Manuela Cempini Valentina Della Porta Susanna Monti