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)

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 B

Chrome tanned leather (CL)

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

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

A

FTIR spectra of dried skin powder spiked with 10-20-30% SCP after esane extraction

-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

CL+ 10% SCP

CL+ 20% SCP

CL+ 30% SCP

SCP/CP 500 mg in 5 mL MeOH(100 mg/mL)

Diluted 1:1 MilliQ(SCP/CP 50 mg/mL)(emulsion)

Gelatin A / Gelatin B /CL(100 mg + 1 mL emulsion)

1:1 MeOH/MilliQemulsion

Esane extraction

Esane extraction

drying

drying

FTIR analysis

blank2h30’ 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

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

blank

1364

GA+SC

P/MeO

H/H2

O

GA+SC

P/IPA/H2

O

GB+SC

P/MeO

H/H2

O

GB+SC

P/IPA/H2

O

CL+S

CP/M

eOH/H2

O

CL+S

CP/IP

A/H2

O

0.0

0.5

1.0

1.5

2.0

OD

1364

/OD

1630

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

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+SC

P/MeO

H/H2

O

GA+SC

P/iso

prop

anol/H2O

*

GB+SC

P/MeO

H/H2

O

GB+SC

P/iso

prop

anol/H2O

SP+S

CP/M

eOH/H2

O

SP+S

CP/is

opropa

nol/H

2O --

GA+CP

30/M

eOH/H2

O

GA+CP

30/is

opropa

nol/H

2O*

GB+CP

30/M

eOH/H2

O

GB+CP

30/is

opropa

nol/H

2O

SP+C

P30/M

eOH/H2

O

SP+C

P30/i

soprop

anol/H2O --

GA+CP

44/M

eOH/H2

O

GA+CP

44/is

opropa

nol/H

2O*

GB+CP

44/M

eOH/H2

O

GB+CP

44/is

opropa

nol/H

2O

SP+C

P44/M

eOH/H2

O

SP+C

P44/i

soprop

anol/H2O --

GA/MeO

H/H2

O

GA/iso

prop

anol/H2O

GB/MeO

H/H2

O

GB/iso

prop

anol/H2O

SP/M

eOH/H2

O

SP/is

opropa

nol/H

2O0

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+SC

P/MeO

H/H2

O

GA+SC

P/iso

prop

anol/H2O

*

GB+SC

P/MeO

H/H2

O

GB+SC

P/iso

prop

anol/H

2O

SP+S

CP/M

eOH/H2

O

SP+S

CP/is

opropa

nol/H

2O --

GA+CP

30/M

eOH/

H2O

GA+CP

30/is

opro

pano

l/H2O

*

GB+CP

30/M

eOH/H2

O

GB+CP

30/is

opro

pano

l/H2O

SP+C

P30/M

eOH/H2

O

SP+C

P30/i

sopr

opan

ol/H

2O --

GA+CP

44/M

eOH/

H2O

GA+CP

44/is

opro

pano

l/H2O

*

GB+CP

44/M

eOH/H2

O

GB+CP

44/is

opro

pano

l/H2O

SP+C

P44/M

eOH/H2

O

SP+C

P44/i

sopr

opan

ol/H

2O --

GA/MeO

H/H2

O

GA/iso

prop

anol/H

2O

GB/MeO

H/H2

O

GB/iso

prop

anol/H2O

SP/M

eOH/

H2O

SP/is

opropa

nol/H

2O

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

--

GA+CP

30/M

eOH/H2

O

GA+CP

30/IP

A/H2

O

GB+CP

30/M

eOH/H2

O

GB+CP

30/IP

A/H2

O

CL+C

P30/M

eOH/H2

O

CL+C

P30/I

PA/H2O --

GA+CP

44/M

eOH/H2

O

GA+CP

44/IP

A/H2

O

GB+CP

44/M

eOH/H2

O

GB+CP

44/IP

A/H2

O

CL+C

P44/M

eOH/H2

O

CL+C

P44/I

PA/H2O0

5

10

15

20

25

30

35

40

7

14-5

40 c

m-1

pea

k ar

ea

CP30 CP44

A B CL A B CL

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