|
MONTEGEN as a scientific education provider........MONTEGEN is involved in a project to support and foster the education of
researchers in the techniques of protein biochemistry, protein structure analysis, and proteomics My personal experience in the area of peptide / protein sequencing analysis is derived by the elucidation structure of the following compounds:  Kassinin [Glu-OMe] Litorin Hylambatin Sauvagine I and Sauvagine II Dermorphin and [Hyp 6 ]Dermorphin Tryptophyllins Protamine Myoglobin Low Molecular Weight immunoregulins (immunosuppressive factors, immune enhancer factor), LH release-inhibiting factor (LHRIF)
I am indebted to prof. Ada Anastasi, prof. Vittorio Erspamer and prof. Agnes Edman Henschen for their useful suggestions and comments. May thanks also to prof. Gerhard Braunitzer for stimulating discussions.
Purification of peptides and proteins from micro- to macro-level |
Methods |
- counter current distribution (CCD)
- affinity chromatography
- displacement chromatogry
- gel chromatography
- ion - exchange chromatography
- fast protein liquid chromatography (FPLC)
- high performance liquid chromatography (HPLC)
- microbore HPLC
- precipitation with alcohol
- precipitation with acetone
- high voltage paper electrophoresis (HVPE)
- low voltage electrophoresis
- polyacrylamide electrophoresis (2D gel electrophoretic system)
- isoelecric focusing
- pseudo titration curves of polypeptides (electrophoresis and isoelectric focusing)
- thin layer chromatography
and paper chromatography
|
Enzymatic degradation of peptides and proteins |
N-Teminus Analysis |
Aminopeptidase M ( EC 3.4.11.2) (AP-M); aminopeptidase M and the diketopiperazine formation:
Phe-Pro-Pro-Trp under the action of AP-M gives origin to Phe and Trp with the diketopiperazine (Pro-Pro) Glu-Lys-Pro-Tyr-..... under the action of AP-M gives origin to Glu, Tyr, and the diketopiperazine
(Lys-Pro).
L-pyroglutamyl peptide hydrolase (EC 3.4.11.8) |
Enzymatic fragmentation |
Cathepsin C (3.4.14.1) (the enzyme splits the peptide bond ...-Ala-Phe-...)Chymotrypsin (EC 3.4.21.1) Trypsin (EC 3.4.21.4) Pronase Arginine-directed
trypsin cleavage (it was obtained by first blocking the epsilon amino groups of the lysines with citraconic anhydride) Specific enzymatic cleavage of the peptide bond ...-Pro-Xxx-...
Splitting the bond ...-Glu-Lys-... by Staphylococcus aureus protease; the ckeavage ability is correlated to the position of the two amino acid residues in the amino acid chain
Subtilisin digestion: specific action at the bonds
3H)-Xxx
Thr-Xxx
|
Specific enzymatc treatment |
Enzymatic desulphatation |
C-Terminus analysis |
Carboxypeptidase A (EC 3.4.12.2)Carboxypeptidase Y (EC 3.4.12.-) |
Diastereoisomers |
D-amino acid oxidase (EC 1.4.3.3.)L-amino acid oxidase |
Degradation by subtilisin of caerulein and analogues. The arrows indicate the cleaved bonds (Tyr *
or Y* = tyrosine-O-sulphate)(Reference:
Montecucchi, P.C. and Gozzini, L. (1982) : Purification and microsequence analysis of active peptides from
amphibian skins - in: "Methods in Protein Sequence Analysis" (Elzinga, M., ed.), Humana Press, Clifton, N.J. U.S.A.), pp 571-572) 
Subtilisin (protease type VIII, Sigma - 0.5M NH 4
OH, pH 8.2, 37 C , 4 h, E/S = 1/50) cleaves the tryptophyl bonds in caeruleins and analogues; in addition it is able to split the linkage Tyr
* -Xxx and Thr - Xxx, exclusively when they occupy the 4th position from N-terminus. Beta-Asp 3
- Caerulein possesses a cross-link between two peptidic segments (segment 1: Z Q D; segment 2: Y*
T G W M D F-NH2); consequently the bond Tyr*-Thr
occuypies an abnormal position and it is not split. The same results are obtained on the desulphated peptides.
Chemical degradation methodsChemical modification of peptides and proteins |
Amino acid analysis with specific reference to |
Methionine, Methionine sulfone, Methionine sulfoxide, Tryptophan, Proline, 3-Hydroxyproline, 4-Hydroxyproline, Tyrosine sulphate, Homoserine,
Homoserine lactone
- Preparation of peptides / proteins for hydrolysis
- Acid / Basic hydrolysis of proteins
- The use of D-amino acid oxidase, L-amino acid oxidase
- About hydrolysis for Trp detection (to assess the Tryptophan content in peptides / proteins :
- (i) samples for amino acid analyses were hydrolysed for 16 h at 110 C in 4 N methanesulphonic acid [containing 0.2% 3-(2aminoethyl)indole] [comparison with an identical sample submitted to hydrolysis
with 6N HCl (constant boiling and containing 0.2% phenol)]. L-Norleucine and 3-nitro-L-tyrosine were used as internal standards. Detection with ninhydrin, OPA / FMOC derivatives
- (ii) Spectrophotometric titration of the tryptophyl residues [ H. Edelhoch Biochemistry 6, 1948 - 1954, (1967)]
Complete enzymatic hydrolysis of proteins (with the use of soluble and insoluble enzymes; treatment with aminopeptidase M and carboxypeptidases to
demonstrate that all the amino acid residues were in the L-form within the structure)
Peptide / protein material visualized with
- Ninhydrin reagent (0,1% ninhydrin in 95% ethanol containing 2% sym-collidine)
- Cl 2 (followed by spraying with 0.5% starch in 0.5% KI) [H.N. Rydon & P.W.G. Smith Nature 169, 922 - 923 (1952)]
- Specific color-reactions for amino acids
- p-dimethylaminobenzaldehyde (Ehrlich's reagent) for tryptophan
- Pauli 's reagent for histidine
- isatin reagent for proline [J. Smith Nature 171, 43 - 44 (1953)]
- isatin reagent followed by the Ehrlich's reagent for 4-hydroxyproline [ C.E. Dangliesh Biochem J. 52, 3 - 14 (1952); J.B. Jepson & I. Smith Nature 172
, 1100 - 1101 (1953); T. Nakajima & B.E. Volcani Science 164 , 1400 - 1401 (1969)]; characterization of 3-hydroxyproline
from 4-hydroxyproline
Sakaguchi's reagent for arginine [J.B. Jepson & I. Smith Nature 172, 1100- 1101 (1953)]
a-nitroso-
b-naphtol reagent for tyrosine [ C.W. Easley
Biochim. Biophys. Acta 107, 386 - 388 (1965)]
platinic-iodide reagent for methionine[ G. Toennies & J.J. Kolb Anal. Chem. 23, 823 - 826 (1951)]
Fluorescamine (0,0002% fluorescamine in acetone) [J. Vanderkerckhove & M. Van Montagu European J. Biochem 44, 279 - 288 (1974)]
Silver stain (protein maps on 2D polyacrylamide gels )
Coomassie Brilliant Blue (CBB) G-250 and R-250 [
Coomassie G (green)-250 is greenish, background is minimized); Coomassie R (red)-250 dye is reddish, high background]
Bromocresol green
The o-phthalaldehyde (OPA) derivatives of the amino acids
The 9-fluorenylmethyloxycarbonyl chloride (FMOC) derivatives of the amino acids
Spectrophotometric titration of the tyrosyl residues [ T. Flatmark Acta Chem. Scand. 18, 1796 - 1798 (1964)]
Separation of amino acids on 2D HVPE (high voltage paper electrophoresis) / chromatography, thin layer chromatography, chromatography on polyamide sheets )
Analysis and calculation (amino acid analysis & sequence analysis)
Treatment with diazonium salts to block the tyrosine residues [Bertaccini G. et al Brit. J. Pharmacol. 25, 363 - 379 (1965)]
Dansyl aminoacids derivatives (separation on polyamide shetts (5 cm x 5 cm)
DABTH amino acids - Separation on polyamide sheets, RP-HPLC
|
End-group methods |
Sequential degradation and determination of amino-terminal residues [
manual Edman degradation , automatic Edman degratation, RP-HPLC systems for PTH-AA analysis ( "N-terminal sequence of some ribosome-inactivating proteins" by
P.C. Montecucchi et al, Int. J. Peptide Protein Res. 33, 263 - 267 (1989)]
- U.V. spectrum of phenylthiohydantoins (from 340 nm to 200 nm): micro moles PTH = (a / b) x K x A 269
[a = total volume of the sample: b = volume used for the spectrum; K = constant specific for each PTH amino acid (PTH Ala = 0,188; PTH Gly = 0,201,..)]
- Dansylation (dansyl, 5-dimethylaminonaphthalene-1-sulphonyl)
- DABITC / PITC-double coupling method
- Removal of the N-terminal amino acid residues by the action of leucine amino-peptidase, amino-peptidase M
- Anomalous degradation from the N-terminal end of peptides with amino-peptidase M: diketopiperazine formation (the removal of the N-terminal amino acid residue is followed by the simultaneous elimination of the
adjacent dipeptide by diketopiperazine formation
- L-pyroglutamyl-peptide hydrolase ( the bond <Glu - Pro-... is resistant to the enzymatic treatment )
- De-blocking peptides [ <Glu - Pro-...] using partial acid hydrolysis (0.01M HCl at 100 C for 6 hr); the peptide was then submitted to Edman degradation
- The N-terminus <Glu - Pro-... : treatment with a proline specific endopeptidase (from Seikagaku Kogyo CO. Ltd., Japan), for example in the structure elucidation of eledoisin
<Glu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2
- Determination of COOH-terminal sequences
- by enzymatic methods (carboxypeptidase A, B and Y )
- by chemical methods [hydrazinolysis, partial acid hydrolysis (with 0.03N HCl at 100 C for 6h), hydrazynolysis after partial acid hydrolysis]
- by isolation of the C-terminal amide fragments (Met-NH2 , Val-NH2), their conversion into the fluorescent dansyl derivatives and subsequent identification by thin - layer
chromatography on polyamide sheets , after extraction with ethyl acetate / water mixtures as described by Tatemoto and Mutt on Proc. Natl. Acad. Sci. U.S.A. 75, 4115 - 4119 (1978);
- by field desorption mass spectrophotometric analysis (Molecular and quasi-molecular ion peaks were always obtained, often accompanied by the MNa + peak) ;
- by electrophoretic migration (HVPE. High voltage Paper Electrophoresis, isoelectric focusing technique)
|
Chemical cleavage of peptides and proteins |
- Cyanogen bromide cleavage (cleavage of the bond ...-Trp-Pro-...by CNBr in formic acid)
- Tyrosine - directed cleavage (with N-bromosuccinimide)
- Tyrosine nitration with tetranitromethane (TNM) [to identify Tyr and Ty(SO3H) residues in protein sequences]
- Arginine - directed trypsin cleavage (with citraconic anhydride)
- Partial acid hydrolysis (PAH) [0.25M acetic acid or 0.01M HCl at 100 C for 10 hr); examples: a) ..-Asp-... [..-Leu-Leu-Leu-Asp-Thr-NH2
giving origin , after PAH, to the three fragments: Leu-Leu-Leu , Asp, Thr-NH2] ; b) ..-Pro-... [ <Glu-Pro-Trp-Val-... giving origin , after PAH, to the fragments: <Glu and Pro-Trp-Val-...]
|
Chemical modifications of proteins |
- Tyrosine nitration with tetranitromethane (TNM) [to identify Tyr and Ty(SO3H) residues in protein sequences]
- The influence of thiol blocking on the resolution of basic proteins by two-dimensional electrophoresis
- Treatment with performic acid [1 ml 30% H2O2
and 9 ml 88% formic acid at room temperature for 1 hour and then the solution will be kept in a refrigerator; preparation of 88% formic acid: 8.9 ml 99% formic acid to 10 ml with distilled water)
- Ethyl ether (peroxide free): Peroxides can be removed from organic compounds by passing the solvent through a column of activated alumina. Test with 1% KI in water in the presence of starch
- Preparation of an oxidized derivative of a methionine-containg peptide: [MetO17] Sauvagine I was obtained by treatment of the standard sample through mild oxidation [0.18% hydrogen peroxide in 0.05 M
acetic acid, 15 min at room temperature, peptide concentration about 0.5 mg/ml as reported by Rusconi L. and Montecucchi, P.C. J. Chromatogr. 346, 390 - 395 (1985)]
|
Methionine sulphoxide form of peptides |
Mild oxidation (0,18% hydrogen peroxide in 0.05 M acetic acid, 15 min. at room temperature; peptide concentration about 0.5 mg /ml)o |
Colorimetric determination of proteins |
Folin - Ciocalteu reagent as reported on J. Bio. Chem. 193, 265 (1951) |
Diastereoisomers separation |
- thin layer chromatography
- column of the amino acid analyzer (for diastereoisomeric dipeptides)
|
Low voltage electrophoresisCellulose acetate electrophoresis |
Low voltage electrophoresis on cellulose acetate and polyamide sheets at pH 8.5. Detection of large peptides and proteins with bromocr4esol green |
Low voltage electrophoresis |
Cellulose acetate electrophoresisLow voltage electrophoresis on cellulose acetate strips (160 x 25
mm) and polyamide sheets at pH 8.0 (0.3 M boric acid adjusted to pH with NaOH, 250V for 90 min, at room temperature). Detection of large peptides and proteins by staining for 30 min with 0,02 % bromocresol
green in distilled water brought to pH 3.5 with glacial acetic acid. Background stain was removed by repeated rinsing with 7.5% acetic acid containing 5% methanol. The strips were successively allowed to dry at room
temperature and then exposed to NH4OH vapours according to the method of Franglen [Franglen, G.T. J. Clin. Pathol. 6, 183 - 186 (1953)]. |
Polyacrylamide gel electrophoresis (PAGE)Electrophoresis in sodium dodecyl sulphate (SDS-PAGE
Analytical and preparative isoelectric focusingf |
Electrophoretic buffers for high-voltage paper electrophoresis HVPE (analytical / preparative) |
pH 1.2 |
99% formic acid / glacial acertic acid / water (170:100:730 by vol.) (1500V / 90 min; 35V x cm -1 , 100-110 mA) |
pH 1.9 |
88% formic acid / glacial acetic acid / waer (5: 78 : 897 by vol.) |
pH 2.7 |
pyridine / glacial acetic acid / water (1:100:899 by vol.) ( for the separation of alpha- and beta- aspartyl peptides) |
pH 5.8 |
pyridine / acetic acid / water (90:10:900 by vol) (1400V / 45 min; 32.5V x cm -1 , 50 mA) |
pH 6.5 |
pyridine / glacial acetic acid / water (50:2:948 by vol.) (1500V / 45 min; 35V x cm -1 , 50 mA) |
The products were characterized by their mobilities at pH 1.2 relative to Glu (E 1.2), at pH 1.9 relative to Glu (E 1.9 ) or cysteic acid ( E
1.9 ) , at pH 5.8 relative to His (E 5.8) or Glu ( E 5.8 ), and at pH 6.58 relative to His (E 6.5) or Glu (E 6.5 ) , according to the migration direction.
Elution of the spots: by electro-elution, with 50% ethanol in water, by chromatography |
Reference: S. Guttmann & R.A. Boissonnas Helv. Chim. Acta 41, 1852 - 1882 (1958) |
Fingerprint technique |
The high voltage paper electrophoresis (HVPE) was run at pH 1.2 in the first direction at 1500 V for 90 min. The electrophoretically separated zones were
further purified by ascending chromatography in the second direction using the solvent system n-butanol / pyridine / glacial acetic acid / water (4:1:1:1 by vol.). The spots were visualized for elution generally by
staining with 0.0002% fluorescamine in acetone. The elution of the spots was made with 50% ethanol |
Chromatography eluent for peptide separation on paper and silica gel thin-layer (analytical / preparative) |
n-butanol / pyridine/ acetic acid / water (4:1:1:1 by vol.)n-butanol / glacial acetic acid / water (4:1:1 by vol.)
n-butanol/ diethylamine / water (4:1:1 by vol.) The chromatographic mobility was calculated in relation to specific amino acids Elution of the spots with 50% ethanol in water. |
Fingerprint (Peptide fragments Map)
HVPE at pH 6.8
Edman manual degradationmicrosequensingusing Reacti-Vials (1 ml) with mininert valves (from Pierce) conveniently modified (Reference:
Montecucchi, P.C. and Gozzini, L. (1982) : Purification and microsequence analysis of active peptides from
amphibian skins - in: "Methods in Protein Sequence Analysis" (Elzinga, M., ed.), Humana Press, Clifton, N.J. U.S.A.), pp 571-572) 
Improvement in the Gas-Phase Sequencer 
The container for TFA
Manometers for the reagents / solvents pressure measure
The gas-phase sequencer [Mod. 470A - Applied Biosystems (Foster City , CA, U.S.A.)] was fitted with a miniaturized conversion flask CuIn the The container for anhydrous trifluoroacetic (TFA) acid was suitably modified from that
purchased from Applied Biosystems Co. : in the new design (appropriate for 40 ml) a major surface is offered for R-3
vapours. In fact the delivery line for R-3 reagent extends into the headspace of the researvoir but not beneath the surface
of the liquid (0.3 cm about the surface). In addition, the manometers to measure the pressure (0 - 5 psi) of the reagents
and solvents used in the sequencer were replaced with those supplied by the Germany WIKA Co. (Milan, Italy), the Italian subsidiary of Alexander Wiegand GmbH & Co. (Germany) (Reference: UK Patent Application GB 2 194948 A -
Application published 23 Mar 1988).
RP-HPLC systems for PTH-AA analysis |
| |
1 |
2 |
3 |
4 |
5 |
System supports |
LiChrosorb |
LiChrospher/60 |
|
Zorbax Bio |
|
Bonded phase |
RP-18 |
CH-8/IIsuper |
cyano |
|
RP-C 18 |
Particle size |
5 |
3-4 |
5 |
5 |
5 |
Column |
|
|
|
|
|
Length (cm) |
25 |
25 |
25 |
25 |
22 |
Int. diam. ( cm) |
0.4 |
0.4 |
0.45 |
0.46 |
0.21 |
Source |
Merck |
Merck |
IBM Instr. |
Du Pont |
ABI |
Conditions |
|
|
|
|
|
Temperature (C) |
62 |
61 |
37 |
35 |
55 |
Flow rate (mL/min) |
1.5 |
2 |
1 |
1.4 |
0.2 |
U.v. detection (nm) |
254 / 320 |
269 / 320 |
269 / 320 |
269 |
269 |
Eluant |
|
|
|
|
|
Buffer A |
10 mM NaOAc |
21 mM NaOAc |
30 mM NaOAc with 5% THF |
6 mM H3PO4 / CH3CN / THF 66:18:16 by vol. (+ 0.001% DTT) |
100 mM NaOAc with 5% THF in water |
pH |
5.2 (AcOH) |
4.9 (AcOH) |
6.27 (AcOH) |
3.15 (NaOH) |
3.6 |
Buffer B |
CH3CN with 0.5% DCE |
CH3CN with 0.5% DCE |
400 mL buffer A to 1000 mL with CH3CN |
|
CH3CN with 500 nmol DMPTU per liter |
System |
isocratic32.5% B |
isocratic32.5% B |
gradient:10-65% B, 12 min 65-80% B, 1 min 80-10%B, 1min 10%B, 10 min |
isocratic |
gradient:10% B, 9.5 min 10-14% B, 1.5 min 14-15% B, 0.3 min 15-40% B, 17 min 40% B, 7 min 40-60% B, 0.1 min 60% B, 8.9 min
60-10%B, 0.5 min |
Sample |
20 microL, containing 50 picomol of each PTH aminoacids (attenuation 2) |
20 microL, containing 50 picomol of each PTH aminoacids (attenuation 2) |
20 microL, containing 50 picomol of each PTH aminoacids (attenuation 2) |
20 microL, containing 50 picomol of each PTH aminoacids (attenuation 2) |
50 microL, containing 10 picomol of each PTH aminoacids (range aufs 0.020, attenuation 4) |
Apparatus |
A Hewlett Packard 1082B HPLC with Hewlett Packard Programmer 85 and Detector 1040 were used. |
A Hewlett Packard 1082B HPLC with Hewlett Packard Programmer 85 and Detector 1040 were used |
A Hewlett Packard 1082B HPLC with Hewlett Packard Programmer 85 and Detector 1040 were used |
A Hewlett Packard 1082B HPLC with Hewlett Packard Programmer 85 and Detector 1040 were used |
The HPLC apparatus (Mod. 120) from Applied Biosystems (Foster City, CA, U.S.A.) has been connected on-line with the Applied Biosystem sequencer
(Mod. 470A. modified) (sample loop: 50 microL; flow cell: 12 microL) , |
Sensitivity |
5 picomol |
5 picomol |
5 picomol |
5 picomol |
5 picomol |
Abbreviations:AcOH, glacial acetic acid; DTT, dithiothreitol; THF, tetrahydrofuran; DMPTU, dimethylphenylthiourea; DCE, dichloroethane; PTH,
phenylthiohydantoin dertivative System 1: a 320-nm window between 4.2 and 6 min has been opened in the detector for the detection of PTH-dehydro Ser (4.76 min) and PTH-dehydro Thr (5.41 min)
System 3: PTH-Asp and PTH-Glu have been identified as methyl ester derivatives System 4: very useful to identify PTH-Lys System 5: PTH-Ser and PTH-Thr were identified also by considering their respective
DTT-trapped derivatives (DTT-PTH dehydroalanine from Ser and DTT-PTH dehydro alpha aminobutyric acid from Thr) which elute between PTH-His and Pft-Tyr. |
Application of Field Desorption (FD) Mass SpectrometryThe sensitivity of the method is estimated approximately 10 -6 to 10-7 g. As reported in the Table, only the peak correspondiong to the molecular weight is obtained for the peptides without any free amino group.
Peptide |
MW |
M+ |
MH+ |
MNa+ |
dermorphin |
802 |
|
803 |
825 |
Hyp6 - dermorphin |
818 |
|
819 |
841 |
deamidated dermorphin |
803 |
|
804 |
826 |
cyclo(-L-Phe - L-Leu-) |
260 |
260 |
|
|
cyclo(-L-Tyr - L-Pro-) |
260 |
260 |
|
|
cyclo(-L-Lys - L-Trp-) |
314 |
314 |
315 |
|
Pyr-Pro-Trp-Val amide |
510 |
510 |
|
|
Met-Asp-Phe- amide |
410 |
|
411 |
|
Pro-Val amide |
213 |
213 |
214 |
|
Pro-Ser amide |
201 |
201 |
202 |
|
Identified post-translational modifications in natural peptidesOne gene can give origine to more amino acid chains |
Identified Modification |
Peptides |
Pyr (pyroglutamic acid) |
The majority of peptides from amphibian skins (caeruleins, sauvagine,...) |
Tyr (SO3)H |
Caeruleins |
4-trans-Hyp |
Dermorphins |
D-Ala |
Dermorphins |
Glu (O-Me) |
Litorins |
AMINO ACID SEQUENCE OF DERMORPHIN
Tyr-ala-Phe-Gly-Tyr-Pro-Ser-NH2 ala= D-AlanineThe first example of a Vertebrate peptide containing a D-aminoacid in its structure
and extracted from the skin of the neotropical frog Phyllomedusa sauvagei - Dermorphin, a D-alanine-containing peptide, with potent opiate-like activity, has been
isolated from skin of the frog Phyllomedusa sauvagei. Complementary DNA (cDNA) libraries (Richter, K., Egger, R. & Kreil, G. (1987) D-alanine in the frog skin peptide dermorphin is derived from L-alanine precursor.
Science 238, 200–202) were constructed from frog skin messenger RNA and screened with a mixture of oligonucleotides that contained the codons complementary to five amino acids of dermorphin. Clones were
detected with inserts coding for different dermorphin precursors. The predicted amino acid sequences of these precursors contained homologous repeats of 35 amino acids that included one copy of the heptapeptide
dermorphin. In these cloned cDNAs, the alanine codon GCG occurred at the position where D-alanine is present in the end product. This suggests the existence of a novel post-translational reaction for the conversion of
an L-amino acid to its D-isomer .A D-amino acid residue present in a D-amino acid-containing peptide is now believed to be due to enzymatically active isomerization
by catalysis of a peptidyl isomerase, by which progression in isomerization is dependent on the amino acid type and its surrounding amino acid sequence. These findings strongly suggest
that enzymatic isomerization must be involved on a peptide synthesized by a ribosomal dependent mechanism ( Shikata, Y., Watanabe, T., Teramoto, T., Inoue, A., Kawakami, Y., Nishizawa, Y., Katayama, K. & Kuwada, M.
(1995) Isolation and characterization of a peptidyl isomerase from funnel web spider venom. J. Biol. Chem. 270, 16719–16723 ; Heck, S.D., Faraci, S., Kelbaugh, P.R., Saccomano, N.A., Thadeio, P.F. &
Volkmann, R.A. (1996) Posttranslational amino acid epimerization: enzyme-catalyzed isomerization of amino acid resudues in peptide chains. Proc. Natl Acad. Sci. 93, 4036–4039). Nevertheless,
the enzyme responsible for the catalyzed isomerization, as postulated, has not been isolated until now from Phyllomedusa sauvagei and Phyllomedusa rhodei. In addition the conversion recovery should
be 100% In fact no traces of the dermorphin analogue with all aminoi acids in the L- configuration was detected in the skin extracts. Consequently, the biosynthesis of dermorphin by a ribosomal dependent
mechanism does not excluded that the incorporation of D-ala into the dermorphin sequence as a consequence of a reaction not sterically specific of the activated aminoacid (in this case D-Ala) with the
correspondent tRNA Ala. In facy the following reactions of the ribosomal protein synthesis:
- aminoacylation of tRNA
- formation of the ternary complex EF-Tu-GTP
- ribosomal binding
- peptide-bond formation
show a certain degree of stereoselectivity, but inferior to that generally accepted in the enzymatic catalysis. |
The MONTEGEN FORUM is open 
|
|
