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Progetto : LIFE10 ENV/IT/000364 «ECOFATTING»

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

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Progetto : LIFE10 ENV/IT/000364 «ECOFATTING»

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  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 • Characterizationoffatagents (solfochloroparaffin, SCP; Chloroparaffins CP30, CP44) • Studyof the interactionoffatagentswithskinproteins (collagen, gelatin, skinpowder)

  3. FTIR spectraof CP, CP30 (C18) CP44 (C14-C17) -SO2- S=O stretching absorptions C-Cl absorptions 1158 608 1365 652 521 564 586 SCP 2926 CP30 2924 2933 609 2855 1446 727 CP44 2855 A 2863 658 1459 740 1262 1461 791 SCP 691 903 CP44 1263 3000 2900 2800 2700 cm-1 1379 1378 621 1123 898 1123 1740 CP30 1741 1745 1800 1600 1400 1200 1000 800 600 500 cm-1

  4. Comparisonbetween FTIR spectraofparaffin (C18-C20), CP30 and CP44 652-607 peak 2926 2916 608 CP30 2855 2849 1261 peak 2933 A 652 2957 P CP44 609 3040 2960 2800 658 1459 1446 CP44 740 A 791 1262 903 727 1466 1379 1263 721 1378 1123 898 CP30 1123 1377 889 Paraffin 1120 1500 1400 1300 1200 1100 1000 900 800 700 600 500 cm-1

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

  6. 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

  8. Collagen CollagenMicrofibrilSegment 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 67 nm Collagen fibril 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. 10-300 nm

  9. METHODS Infraredspectroscopy (ATR-FTIR) Computationalstudy ThermogravimetricAnalysis (TGA)

  10. Is gelatin a good model? FTIR spectra of gelatins and skin powder Amide II 1635 1629 Amide I 1523 Gelatin B Gelatin Merck 1538 1442 1237 1398 1079 1330 1028 1161 1447 1236 1400 1334 A 1080 1030 972 Skinpowder 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 cm-1

  11. FTIR spectra of dried skin powder spiked with 10-20-30% SCP 1363 1159 2924 1633 SP+ 30% SCP 1540 1457 2854 A SP+ 20% SCP 1237 586 564 529 3285 1080 SP SP+ 10% SCP 1031 1364 690 3073 1157 1463 851 SCP 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 cm-1

  12. FTIR spectra of dried skin powder spiked with 10-20-30% SCP after esane extraction 1633 1532 1448 1367 2927 1232 SP+ 20% SCP 1159 A 3283 2859 SP+ 30% SCP 3067 1364 1080 SP+ 10% SCP SP 2854 1157 2924 1463 691 852 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 cm-1

  13. New protocoltoimprove sample homogeneity: solubilizationof SCP and CP in MeOH or isopropanol and treatment ofgelatinswith the MeOH/H2O or isopropanol/H2O emulsion SCP/CP 500 mg in 5 mLMeOH (100 mg/mL) 1:1 MeOH/MilliQ emulsion Diulted 1:1 MilliQ (SCP/CP 50 mg/mL) (emulsion) Gelatin A and B (Sigma) (100 mg + 1 mLemulsion) blank 2h30’ incubationtime RT and stirring pH monitoring and adjustment* Esaneextraction Esaneextraction drying drying FTIR analysis TGA analysis *pHchanged and wasadjustedonly in the case ofGelatin A treatedwith SCP in isopropanol/H2O emulsion

  14. FTIR spectra of Gelatin B + 50% CP30 (C18) /CP44 (C14-C17) MeOH/water emulsion after esane extraction and drying 2925 2926 2856 1458 CP30 A 2860 3292 1629 1633 610 3600 3200 2800 1547 1378 1541 1237 (1263 cm-1 in pure CP) A 657 1453 1310 1378 1239 CP44 726 1338 1203 1081 563 1034 591 529 1742 971 899 936 blank 1160 1033 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 cm-1

  15. FTIR spectra of Gelatin B + 50% SCP in MeOH/water emulsion after esane extraction and drying 1364 1159 SCP 564 2925 530 586 2934 1459 A 1631 2855 1545 1235 2864 691 3297 1080 621 3076 1740 1031 blank 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 cm-1

  16. Semi-quantitative spectroscopic parameters to evaluate SCP/CP-protein interactions: summary of gelatin and skin powder reactivity -SO2- S=O stretching vibrationtypicalof SCP Micelle size??

  17. Semi-quantitative spectroscopic parameters to evaluate SCP/CP-protein interactions: summary of gelatin and skin powder reactivity CH2 stretching vibrationsof SCP and CP SCP CP44 CP30 blank A B SP

  18. Semi-quantitative spectroscopic parameters to evaluate SCP/CP-protein interactions: summary of gelatin and skin powder reactivity CH2 bendingvibrationsof SCP and CP SCP CP44 CP30 blank

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

  20. FTIR spectra of dried skin powder + 50% SCP in MeOH or isopropanol after esane extraction and drying 587 534 1157 2924 1364 2854 691 564 520 1463 623 SCP 524 1635 1400 1538 1447 1236 1334 1203 1080 972 3283 2931 1163 1030 3070 SP 523 1236 1159 1629 A 1368 1033 1542 2932 1449 1201 3286 1336 1079 3075 984 2875 SP+SCP in MeOH 521 1630 1540 1235 1030 1447 1335 1398 1204 3285 945 1079 3073 2933 1161 972 SP+SCP in Isopr 4000,0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450,0 cm-1

  21. FTIR spectra of dried skin powder + 50% CP44 in MeOH or isopropanol after esane extraction and drying 608 1262 903 740 2933 652 1446 984 791 1123 2863 1379 1056 1740 524 CP44 1635 1236 1400 1538 1080 1447 1334 1203 972 2931 3283 1163 1030 3070 SP 528 1021 554 1237 Amine C-N stretch ??? (1360-1020) Ether C-O stretch?? A 1635 1448 1336 2939 1547 3302 1205 1399 2831 3077 1163 SP+CP44 in MeOH 524 1634 1534 1080 1445 1234 1030 1399 2933 1161 3284 3069 SP+CP44 in Isopr 1334 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 cm-1

  22. FTIR spectra of dried skin powder + 50% CP30 in MeOH or isopropanolafter 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 1631 Amine C-N stretch ??? (1360-1020) Ether C-O stretch?? A 1449 1235 1544 1022 1397 3287 1336 1204 2928 1080 3075 SP+CP30 in MeOH 524 1631 1235 1448 1538 1334 1080 2927 3284 1399 1161 1030 2857 3072 SP+CP30 in Isopr 4000,0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450,0 cm-1

  23. Collagenmodelforcomputationalstudy Collagenmicrofibrilsegment (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 ASNSER 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 ASNSER GLY GLU NME ACE GLY LEU GLN GLY PRO HPR GLY PRO HPR GLY SERHPR GLY GLUGLN GLY PRO SER GLY ALA SER GLY PRO NME ACE GLY LEU GLN GLY LEU HPR GLY LEU ALA GLY HID HID GLY ASPGLN GLY ALA HPR GLY ALA VAL GLY PRO NME ACE GLY LEU GLN GLY PRO HPR GLY PRO HPR GLY SERHPR GLY GLUGLN 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 ARGTHR GLY THRHPR 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 LYSHPR GLY GLUGLN GLY VAL HPR GLY ASP LEU GLY ALA NME ACE GLY PRO ALA GLY THR ALA GLY GLU ALA GLY LYSHPR GLY GLUARG GLY ILE HPR GLY GLU PHE GLY LEU NME ACE GLY PRO ALA GLY PRO HPR GLY GLU ALA GLY LYSHPR GLY GLUGLN 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- T=37°C Sulpho Chlorinated Paraffin SCP_C20. Molecular Electrostatic Potential (MEP) on the solvent accessible surface (red areas: negative potential, blue areas: positive potential).

  24. Collagencomputationalstudy: preliminaryresults 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

  25. Collagencomputationalstudy: preliminaryresults 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. In progress!

  28. 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-ethylbenzyl amine NHR2 + SCP  SCP-NR2 +HCl HPLC/UV/fluorescence

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

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