- Email: [email protected]
[33] Primary structure of insoluble elastin
606
ELASTIN
[33]
[33] P r i m a r y S t r u c t u r e o f I n s o l u b l e E l a s t i n
By RASHID A. ANWAR The problems encountered in the study of the primary structure of mature elastin are attributable to its extreme insolubility and high degree of cross-linking. The protein is insoluble in all nonhydrolytic solvents. For this reason, it is practically impossible to establish homogeneity (or lack of it) of an elastin preparation. The solubilization of elastin by chemical or enzymic means usually generates a complex mixture of peptides. Fortunately, in some cases a soluble precursor of elastin (tropoelastin) can be isolated and its primary structure studied essentially by classical sequencing techniques (see Chapters [36]-[40] on tropoelastin). Nevertheless, the study of mature elastin, especially around the cross-links, was clearly required to understand the structure-function relationship of elastin and the mechanism of formation of the cross-links. These considerations led to the structural studies of solubilized cross-linked peptides of elastin.'-5 In order to determine the amino acid sequences of the cross-linked peptides, the cross-linked peptide chains must be resolved into single-chain peptides, so that the amino acid sequence assignments can be made unambiguously. With the use of Edman degradation, single-chain peptides can be released from the carboxyl groups of the cross-links present in the elastolytic peptides of elastin. These peptides can then be purified and their amino acid sequences can be determined.'
The Rationale behind the Use of the Preparative Edman Degradation for the Release of Carbo~ryl-Terminal Peptides from the Elastolytic Cross-linked Peptides. In earlier work, ~ the release of single-chain peptides from the desmosine-containing elastolytic peptides of elastin was attempted by means of the chemical cleavage of the pyridine rings (periodatepermangnate oxidationT). The characterization of peptides thus released from the desmosine cross-links indicated that elastase was cleaving at and very close to the NH~ terminals of the cross-links. In retrospect, this was ' G. E. Gerber and R. A. Anwar, J. Biol. Chem. 249, 5200 (1974). 2 G. E. Gerber and R. A. Anwar, Biochem. J. 149, 685 (1975). 3 K. M. Baig, M. Vlaovic, and R. A. Anwar, Biochem. J. 185, 611 (1980). 4 j. A. Foster, L. Rubin, H. M. Kagan, C. Franzblau, E. Bruenger, and L. B. Sandberg, J. Biol. Chem. 249, 6191 (1974). R. P. Mecham and J. A. Foster, Biochem J. 173, 617 (1978). 6 W. Shimada, A. Bowman, N. R. Davis, and R. A. Anwar, Biochem. Biophys. Res. Commun. 37, 191 (1969). 7 R. U. Lemieux and E. Von Rudloff, Can. J. Chem. 33, 1701 0956).
METHODSIN ENZYMOLOGY,VOL. 82
Copyright © 1982by Academic Press, Inc. All rights of reproduction in any formreserved. ISBN 0-12-181982-5
[33]
PRIMARY STRUCTURE OF INSOLUBLE ELASTIN
O>jIHN~. ~CH 3
C6H5
T
--.-"~o
/
' o~/"h
.~.~ \c.~
1.J v
- . i ~ ,-.;T. - v
'~ , ~ /NH-P1 H2N f ~ 0
607
NH2 jGH s
' " ~ . ""-P3
....... :
,f.
I.,TC ~ . .
v
/c..
S C-H6 ~ PTH-AIa
-.l~y
v
."~--""-P3
'[. HN'-J~ I ~-O S//[__IN/
Released Peptides
C6H5 FIG. 1. The use of the Edman degradation for the release of carboxyl-terminal peptides from the elastolytic, desmosine-containing, cross-linked peptides of elastin. PITC, phenylisothiocyanate; PTH, phenylthiohydantoin.
expected because the purified elastase has a strong specificity for alanyl peptide bonds and the cross-linked regions of elastin are very rich in alanine content. The amino-terminal analysis of the elastolytic crosslinked peptides of elastin confirmed the presence of free a-amino groups of desmosines and isodesmosines in these peptides. It was, therefore, thought possible to release carboxyl-terminal peptides from the elastolytic, desmo sine- and isodesmosine-containingpeptides by Edman degradation. 1 Figure 1 shows the Edman degradation of one of the many possible structures present in the cross-linked peptide mixtures and illustrates the rationale of this approach. The thick dark lines represent the desmosine cross-links. At those positions, where the cross-link itself is the NH2 terminal, the Edman degradation will release the peptides attached to the a-carboxyl groups of the cross-link (P1 in this case). The released singlechain peptides can then be separated from the cross-linked ones on the basis of size. At position P2, after the first cycle of Edman degradation, the amino-terminal alanine will be released as PTH-Ala, with the concomitant liberation of the a-amino group of the cross-link. Therefore, the second cycle of Edman degradation will release P2 from the carboxyl terminal of the cross-link. The peptide Pa will not be released until the cross-link NH2 terminal immediately preceding Pa becomes available. Thus, P3 will be released after four cycles of Edman degradation. It is apparent that after each cycle of Edman degradation the released singlechain peptides must be separated from the cross-linked peptides.
608
ELASTIN
[33]
Procedures
Solubilization and Exhaustive Digestion of Elastin with Porcine Pancreatic Elastase. It is important to use elastase purified to homogeneity. 8 Partially purified elastase preparations contain carboxypeptidase A and other proteolytic activities. Therefore, the purity of commercially obtained elastase should be ascertained before use (see Chapter [32]). All buffers are saturated with chloroform to minimize bacterial contamination. Elastin powder (10 g) is suspended in 1 liter of 0.01 N NH4HCO3 buffer, pH 8.8, and stirred overnight at room temperature. The next morning, 20 mg of elastase are added, and the mixture is allowed to incubate at room temperature (22-24°). After 24 hr of incubation another 20 mg of elastase are added, and the incubation is allowed to continue for additional 24 hr. The pH of the incubation mixture is maintained between 8.7 and 8.9 with the addition of concentrated NH4OH. The incubation mixture is then concentrated to 150 ml, under nitrogen pressure (80-90 psi) in a Diaflo apparatus (Amicon) equipped with a UM-10 membrane. The retentate is diluted with 350 ml of 0.01 N NHaHCO3 buffer, pH 8.8, and reconcentrated to 150 ml; this procedure is repeated two more times. The desmosine-containing cross-linked peptides do not pass through a UM-I0 membrane and are thus retained. This permits the removal of small elastolytic peptides that inhibit elastase. The retentate is then diluted to 500 ml with the same buffer and further digested with two more additions of elastase (20 mg each). The mixture is allowed to incubate for 24 hr after each addition. Finally, the solution is boiled for 5 min and filtered through a Whatman No. 5 paper. Isolation of Cross-linked Peptides. The pH of the elastase digest containing the cross-linked peptides is adjusted to 4.0 with glacial acetic acid. The digest is then applied to a column of cellulose phosphate (Cellex-P; 2.0 × 30 cm), preequilibrated with 0.01 N sodium acetate buffer, pH 4.5. The column is washed with 500 ml of the preequilibration buffer and then eluted with 0.1 N NaCI in the same buffer. Fractions of 7.5 ml are collected, and their absorbance at 280 nm is measured after suitable dilution when necessary. A typical elution profile is shown in Fig. 2. Under these conditions, over 90% of the desmosine-containing peptides present in an elastase digest of elastin absorb onto a cellulose phosphate (CP) column and are eluted in fractions labeled CPB in Fig. 2. It is useful and desirable first to study the desmosine-containing crosslinked peptides, which are essentially free of other cross-links. This provides unambiguous results about the environment of the desmosines. The desmosine-containing cross-linked peptides essentially free of other 8 A. S. Narayanan and R. A. Anwar, Biochem. J. 114, 11 (1%9).
[33]
609
PRIMARY STRUCTURE OF INSOLUBLE ELASTIN
25
20-
o
oO
15-
10
i
0
CPA I
CPB
~
'
10
I
20
30
Fraction Number FIG. 2. Chromatographic separation of elastolytic peptides of elastin on cellulose phosphate.
cross-links can be obtained by further fractionation of CPB (Fig. 2) on Sephadex G-50 and Sephadex G-25. Fractions labeled CPB are pooled, dried under reduced pressure, and dissolved in 3-5 ml of 0.01 M NH4HCO3 buffer, pH 8.8. This solution is applied on a Sephadex G-50 (fine) column (4 × 80 cm) previously equilibrated with 0.01 M NH4HCOa buffer, pH 8.8. The column is then eluted with the same buffer at a flow
610
ELASTIN
[33]
1.5
CD
1.0
0.5
0
I
I
i
i
I
20
40
60
80
100
Fraction Number FIG. 3. Chromatography of fraction CPB (Fig. 2) on Sephadex G-50.
rate of 100 ml/hr. Fractions of 10 ml are collected, and their absorbance is measured at 280 nm. A typical elution profile of a Sephadex G-50 chromatography is shown in Fig. 3. The fractions are pooled as indicated in Fig. 3. The pooled fractions appearing under the peak marked G-50-III are dried under reduced pressure on a rotary evaporator and dissolved in about 1 ml of 0.01 M NH4HCO3 buffer, pH 8.8. This solution is applied on to a Sephadex G-25 (fine) column (2.0 × 35 cm), previously equilibrated with 0.01 M NH4HCO3 buffer, pH 8.8, and the column is eluted with the same buffer. Fractions of 5.0 ml are collected, and absorbance is measured at 275 nm after suitable dilution, when necessary. A typical elution profile of Sephadex G-25 chromatography is shown in Fig. 4. The desmosine-containing cross-linked peptides present in fractions labeled G-25.2 are free of other cross-links and contain 25-30% of the total desmosine and isodesmosine present in elastin. Before pooling the fractions, it is prudent to analyze (amino acid analysis) suitable aliquots from individual fractions on either end of the peak marked G-25.2 to ascertain that the fractions are essentially free of cross-links other than the desmosines (see Chapters [16] and [31]).
12C
[33]
PRIMARY STRUCTURE OF INSOLUBLE ELASTIN
611
8.0
6.0
4.0
2.0
G25.1 I
0
•5
15
1'0 Fraction
2'0
Number
FIG. 4. C h r o m a t o g r a p h y of fraction G-50-III (Fig. 3) on Sephadex G-25.
The pooled fractions (G-25.2) are dried under reduced pressure on a rotary evaporator and the dried material is dissolved in 1 ml of pyridinewater-acetic acid ( 4 : 4 : 1) mixture and applied on to a Sephadex LH-20 column (1.9 x 60 cm) previously equilibrated with 50% pyridine solution. The column is eluted with 50% pyridine at a flow rate of 28 ml/hr. Fractions o f 3.5 ml are collected, absorbance is measured at 330 nm, and aliquots (10 /zl) are assayed with ninhydrin after alkaline hydrolysis. Owing to the presence of 50% pyridine in elution solvent, absorbance at 280 nm or 275 nm cannot be measured. The excluded material elutes in fractions labeled A in Fig. 5. These fractions, which contain all the desmosine-containing cross-linked peptides applied to the column, are pooled and dried under reduced pressure on a rotary evaporator. This dried material is used for the release of single-chain peptides from the carboxyl terminals of the desmosine cross-links. As an example of the degree of purification expected at each step, amino acid compositions o f crosslinked bovine elastin peptides at various stages of purification are shown in the table.
25
612
ELASTIN
.....
OL§ V
d I
[33]
o
I
1
0
0
.-~
~
< 0
/
d
0 O
E Z
I
0
0
I
I
I 0
0
d
0
OEE y
0
--
0
[33]
613
PRIMARY STRUCTURE OF INSOLUBLE ELASTIN AMINO ACID COMPOSITION OF CROSS-LINKED BOVINE ELASTIN PEPTIDES AT VARIOUS STAGES OF PURIFICATION a
Amino acid
CPB
G-50-I G-50-II G-50-III G-25.1 G-25.2 G-25.3
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Isodesmosine Desmosine Lysinonorleucine
0.2 0.3 0.6 0.5 4.8 10.4 16.8 1.7 0.6 1.6 0.4 1.2 0.41 0.59 0.1 0.9 1.1
0.4 0.4 0.6 0.7 6.6 15.3 19.6 3.7 0.9 2.7 0.7 2.0 0.42 0.58 0.1 0.4 0.5
Lysine
Arginine
0.1 0.3 0.5 0.5 4.4 14.2 17.6 2.3 0.5 1.8 0.6 1.7 0.35 0.65 0.1 0.3 0.4
0.2 0.3 0.4 0.4 1.5 4.5 11.5 0.8 0.3 0.7 0.3 1.1 0.31 0.69 0.1 0.2 0.3
0.1 0.3 0.4 0.3 2.0 5.7 13.0 0.9 0.2 0.9 0.3 1.0 0.34 0.66 0.1 0.1 0.3
0.1 0.2 0.4 0.3 1.4 4.4 11.4 0.7 0.2 0.7 0.3 1.0 0.33 0.67 -0.1 0.3
0.5 0.4 0.9 1.0 3.1 9.2 16.9 1.5 0.7 1.4 0.4 2.9 0.32 0.68
A 0.1 0.2 0.3 0.3 1.6 4.1 11.6 0.6 0.2 0.7 0.3 1.0 0.32 0.68
1.4
--
0.3 1.4
0.1 0.1
Values are expressed as residues per residue of cross-link (i.e., desmosine plus isodesmosine).
Release of Carboxyl-Terminal Peptides and Separation of the Released Single-Chain Peptides from the Cross-linked Ones. T h e dried m a t e r i a l ( 2 0 - 3 0 ~ m o l o f d e s m o s i n e plus i s o d e s m o s i n e ) is dissolved in 4 ml o f c o u p l i n g buffer (50% p y r i d i n e , 2% t r i e t h y l a m i n e ) in a n a c i d - w a s h e d P y r e x test t u b e , a n d the t u b e a n d the c o n t e n t s are flushed w i t h n i t r o g e n . A t least 50-fold e x c e s s o f p h e n y l i s o t h i o c y a n a t e is a d d e d to the s o l u t i o n a n d the m i x t u r e is i n c u b a t e d u n d e r n i t r o g e n at 50° for 45 rain. T h e r e a c t i o n mixture is t h e n dried on a r o t a r y E v a p o - M i x at 60 ° u n d e r r e d u c e d p r e s s u r e . A f t e r d r y i n g , the v a c u u m is r e l e a s e d u n d e r nitrogen. T h i s is a c c o m p l i s h e d with the u s e o f a t h r e e - w a y s t o p c o c k ; o n e e n d leads to the test t u b e , t h r o u g h r o t a r y E v a p o - M i x c o n n e c t i o n s ; the o t h e r e n d to a v a c u u m p u m p ; a n d the third end to a n i t r o g e n t a n k . T h e dried r e a c t i o n m i x t u r e is i n c u b a t e d w i t h 1 ml o f trifluoroacetic acid at 50 ° for 15 m i n , a n d t r i f l u o r o a c e t i c acid is r e m o v e d u n d e r r e d u c e d p r e s s u r e . T h e r e s i d u e is dissolved in 1.0 ml o f p y r i d i n e - w a t e r - a c e t i c acid (4 : 4 : 1) m i x t u r e a n d applied to a S e p h a d e x L H - 2 0 c o l u m n (1.9 x 60 cm) p r e v i o u s l y e q u i l i b r a t e d with 50% p y r i d i n e . T h e c o l u m n is e l u t e d with 50% p y r i d i n e at a flow rate o f 28 ml/hr. F r a c t i o n s o f 3.5 ml are c o l l e c t e d , a b s o r b a n c e is m e a s u r e d at 330 n m , a n d aliquots (10/zl) are a s s a y e d with n i n h y d r i n after alkaline h y d r o l y s i s . T h e c r o s s - l i n k e d p e p t i d e s elute in
614
ELASTIN
[33]
fractions labeled A, and the released single-chain peptides appear in fractions labeled B (Fig. 5). Fractions under A are pooled and dried, and the Edman degradation and Sephadex LH-20 chromatography are repeated. It may be emphasized that after each cycle of Edman degradation the released single-chain peptides (fraction B, Fig. 5) are separated from the cross-linked peptides (fraction A). Five cycles of Edman degradation can be carried out for this purpose without any problem. It may be mentioned that most of the information becomes available after three cycles of degradation. Purification of Released Single-Chain Peptides. The released singlechain peptides obtained after each cycle of Edman degradation may be purified separately and then sequenced, or these peptides from several cycles may be pooled and then purified. The pooling of released peptides from several cycles allows better recovery of low-yield peptides. Several methods of peptide purification have been described2 '~° In author's laboratory 2'3 initial separation is carried out on a column (0.9 × 55 cm) of Beckman AA-15 resin (a substitute for AA-15 resin is W-2 resin) with pyridine acetate buffers (I: 0.05 N pyridine acetate, pH 2.6; II: 0.10 N pyridine acetate, pH 3.15; III: 0.5 N pyridine acetate, pH 3.7; IV: 2.0 M pyridine acetate, pH 5.0). The column is first developed with a gradient consisting of 300 ml of each of buffers I, II, and III connected in sequence in the order of increasing pH and molarity. This is followed by a gradient consisting of 150 ml of buffers III and IV. The column is eluted at 55° and a flow rate of 42 ml/hr. The peptide peaks are detected with ninhydrin after alkaline hydrolysis. Further purification of peptides (when required) is achieved by paper chromatography (solvent: n-butanol-n-butyl acetate-acetic acid-water, 135 : 6 : 30 : 50) and/or high-voltage paper electrophoresis, in 0.25 M pyridine acetate buffer, pH 3.5. Sequencing of Purified Released Peptides. Methods for sequence analysis of purified single chain peptides have been described. H'12 In the author's laboratory, the purified single-chain peptides, after amino acid analysis, are sequenced manually with the use of Edman degradation followed by dansylation, la It may be emphasized that coupling and cyclization are carried out under nitrogen. Dansyl amino acids are identified by chromatography on polyamide sheets. TM Other elastolytic, cross-linked peptides of elastin (G-50-I and G-50-II) may be examined by a procedure identical to that described above for 9 This series, Vol. 25, Section ~0 This series, Vol. 47, Section 11 This series, VoL 25, Section lz This series, Vol. 47, Section 1.~ W. R. Gray, this series, Vol. ~4 W. R. Gray, this series, Vol.
VI. VII. VII. VIII. 25, p. 333. 25, p. 135.
[34]
B I O S Y N T H E S I S OF I N S O L U B L E E L A S T I N
615
fraction G-25.27 The above approach has been successfully used in a comparative study of elastins from different species and tissues. 2 Simultaneous Sequencing of Two Cross-linked Peptide Chains. Foster and co-workers 4"~ have studied desmosine-containing cross-linked peptides isolated from subtilisin and thermolysin digests of elastin. Peptides were purified by chromatography on ion-exchange resin (Technicon peptide resin, 4% cross-linked, or Aminex 50W-X4, or type-P chromobead) with pyridine acetate buffers and by gel filtration (e.g., Sephadex G-50 or Sephadex G-25). In this approach, cross-linked peptide, judged to be homogeneous, is sequenced on a Beckman sequencer. As two or more chains are sequenced simultaneously, the cross-link positions are assigned on the basis of amino acid residue yield obtained at each cycle of degradation. For example, the sequence data (first cycle, Ala, 127 nmol; second cycle, Ala, 85 nmol; third cycle, Ala 61 nmol; fourth cycle, Ala, 81 nmol; fifth cycle, Ala, 34 nmol) are interpreted to show the sequencing of two chains. At first cycle, both chains contain Ala. At second cycle the yield of Ala is about half that of the first cycle; therefore, one chain has Ala at this position, and in the other chain this position is occupied by the crosslink, and the same is true of the third cycle. At the fourth cycle both positions are assigned to Ala, and in the fifth cycle one position is assigned to Ala and the other to cross-link. As the peptides carboxyl terminal to the cross-links are released, the yield drops sharply and the assignments become difficult. Therefore, in a way this approach is complementary to the one described above. It is apparent that the cross-linked peptide under examination must be essentially homogeneous. It may also be pointed out that amino-terminal analysis alone is not a sufficient proof of homogeneity of desmosine crosslinked peptides, because all desmosine cross-links have alanine residues at the amino terminals and thus will show two alanine residues per residue of desmosine (i.e., desmosine plus isodesmosine) whether the peptide is pure or is a mixture of several peptides.
[34] B i o s y n t h e s i s o f I n s o l u b l e E l a s t i n in C e l l a n d Organ Cultures
By
CARL
FRANZBLAU
and
BARBARA
FARIS
The formation of insoluble elastin in in v#ro systems such as cell and organ cultures has been the focus of several laboratories in recent years. As noted in the overall introduction (Chapter [30]) to the chapters on
METHODS IN ENZYMOLOGY, VOL. 82
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181982-5
ELASTIN
[33]
[33] P r i m a r y S t r u c t u r e o f I n s o l u b l e E l a s t i n
By RASHID A. ANWAR The problems encountered in the study of the primary structure of mature elastin are attributable to its extreme insolubility and high degree of cross-linking. The protein is insoluble in all nonhydrolytic solvents. For this reason, it is practically impossible to establish homogeneity (or lack of it) of an elastin preparation. The solubilization of elastin by chemical or enzymic means usually generates a complex mixture of peptides. Fortunately, in some cases a soluble precursor of elastin (tropoelastin) can be isolated and its primary structure studied essentially by classical sequencing techniques (see Chapters [36]-[40] on tropoelastin). Nevertheless, the study of mature elastin, especially around the cross-links, was clearly required to understand the structure-function relationship of elastin and the mechanism of formation of the cross-links. These considerations led to the structural studies of solubilized cross-linked peptides of elastin.'-5 In order to determine the amino acid sequences of the cross-linked peptides, the cross-linked peptide chains must be resolved into single-chain peptides, so that the amino acid sequence assignments can be made unambiguously. With the use of Edman degradation, single-chain peptides can be released from the carboxyl groups of the cross-links present in the elastolytic peptides of elastin. These peptides can then be purified and their amino acid sequences can be determined.'
The Rationale behind the Use of the Preparative Edman Degradation for the Release of Carbo~ryl-Terminal Peptides from the Elastolytic Cross-linked Peptides. In earlier work, ~ the release of single-chain peptides from the desmosine-containing elastolytic peptides of elastin was attempted by means of the chemical cleavage of the pyridine rings (periodatepermangnate oxidationT). The characterization of peptides thus released from the desmosine cross-links indicated that elastase was cleaving at and very close to the NH~ terminals of the cross-links. In retrospect, this was ' G. E. Gerber and R. A. Anwar, J. Biol. Chem. 249, 5200 (1974). 2 G. E. Gerber and R. A. Anwar, Biochem. J. 149, 685 (1975). 3 K. M. Baig, M. Vlaovic, and R. A. Anwar, Biochem. J. 185, 611 (1980). 4 j. A. Foster, L. Rubin, H. M. Kagan, C. Franzblau, E. Bruenger, and L. B. Sandberg, J. Biol. Chem. 249, 6191 (1974). R. P. Mecham and J. A. Foster, Biochem J. 173, 617 (1978). 6 W. Shimada, A. Bowman, N. R. Davis, and R. A. Anwar, Biochem. Biophys. Res. Commun. 37, 191 (1969). 7 R. U. Lemieux and E. Von Rudloff, Can. J. Chem. 33, 1701 0956).
METHODSIN ENZYMOLOGY,VOL. 82
Copyright © 1982by Academic Press, Inc. All rights of reproduction in any formreserved. ISBN 0-12-181982-5
[33]
PRIMARY STRUCTURE OF INSOLUBLE ELASTIN
O>jIHN~. ~CH 3
C6H5
T
--.-"~o
/
' o~/"h
.~.~ \c.~
1.J v
- . i ~ ,-.;T. - v
'~ , ~ /NH-P1 H2N f ~ 0
607
NH2 jGH s
' " ~ . ""-P3
....... :
,f.
I.,TC ~ . .
v
/c..
S C-H6 ~ PTH-AIa
-.l~y
v
."~--""-P3
'[. HN'-J~ I ~-O S//[__IN/
Released Peptides
C6H5 FIG. 1. The use of the Edman degradation for the release of carboxyl-terminal peptides from the elastolytic, desmosine-containing, cross-linked peptides of elastin. PITC, phenylisothiocyanate; PTH, phenylthiohydantoin.
expected because the purified elastase has a strong specificity for alanyl peptide bonds and the cross-linked regions of elastin are very rich in alanine content. The amino-terminal analysis of the elastolytic crosslinked peptides of elastin confirmed the presence of free a-amino groups of desmosines and isodesmosines in these peptides. It was, therefore, thought possible to release carboxyl-terminal peptides from the elastolytic, desmo sine- and isodesmosine-containingpeptides by Edman degradation. 1 Figure 1 shows the Edman degradation of one of the many possible structures present in the cross-linked peptide mixtures and illustrates the rationale of this approach. The thick dark lines represent the desmosine cross-links. At those positions, where the cross-link itself is the NH2 terminal, the Edman degradation will release the peptides attached to the a-carboxyl groups of the cross-link (P1 in this case). The released singlechain peptides can then be separated from the cross-linked ones on the basis of size. At position P2, after the first cycle of Edman degradation, the amino-terminal alanine will be released as PTH-Ala, with the concomitant liberation of the a-amino group of the cross-link. Therefore, the second cycle of Edman degradation will release P2 from the carboxyl terminal of the cross-link. The peptide Pa will not be released until the cross-link NH2 terminal immediately preceding Pa becomes available. Thus, P3 will be released after four cycles of Edman degradation. It is apparent that after each cycle of Edman degradation the released singlechain peptides must be separated from the cross-linked peptides.
608
ELASTIN
[33]
Procedures
Solubilization and Exhaustive Digestion of Elastin with Porcine Pancreatic Elastase. It is important to use elastase purified to homogeneity. 8 Partially purified elastase preparations contain carboxypeptidase A and other proteolytic activities. Therefore, the purity of commercially obtained elastase should be ascertained before use (see Chapter [32]). All buffers are saturated with chloroform to minimize bacterial contamination. Elastin powder (10 g) is suspended in 1 liter of 0.01 N NH4HCO3 buffer, pH 8.8, and stirred overnight at room temperature. The next morning, 20 mg of elastase are added, and the mixture is allowed to incubate at room temperature (22-24°). After 24 hr of incubation another 20 mg of elastase are added, and the incubation is allowed to continue for additional 24 hr. The pH of the incubation mixture is maintained between 8.7 and 8.9 with the addition of concentrated NH4OH. The incubation mixture is then concentrated to 150 ml, under nitrogen pressure (80-90 psi) in a Diaflo apparatus (Amicon) equipped with a UM-10 membrane. The retentate is diluted with 350 ml of 0.01 N NHaHCO3 buffer, pH 8.8, and reconcentrated to 150 ml; this procedure is repeated two more times. The desmosine-containing cross-linked peptides do not pass through a UM-I0 membrane and are thus retained. This permits the removal of small elastolytic peptides that inhibit elastase. The retentate is then diluted to 500 ml with the same buffer and further digested with two more additions of elastase (20 mg each). The mixture is allowed to incubate for 24 hr after each addition. Finally, the solution is boiled for 5 min and filtered through a Whatman No. 5 paper. Isolation of Cross-linked Peptides. The pH of the elastase digest containing the cross-linked peptides is adjusted to 4.0 with glacial acetic acid. The digest is then applied to a column of cellulose phosphate (Cellex-P; 2.0 × 30 cm), preequilibrated with 0.01 N sodium acetate buffer, pH 4.5. The column is washed with 500 ml of the preequilibration buffer and then eluted with 0.1 N NaCI in the same buffer. Fractions of 7.5 ml are collected, and their absorbance at 280 nm is measured after suitable dilution when necessary. A typical elution profile is shown in Fig. 2. Under these conditions, over 90% of the desmosine-containing peptides present in an elastase digest of elastin absorb onto a cellulose phosphate (CP) column and are eluted in fractions labeled CPB in Fig. 2. It is useful and desirable first to study the desmosine-containing crosslinked peptides, which are essentially free of other cross-links. This provides unambiguous results about the environment of the desmosines. The desmosine-containing cross-linked peptides essentially free of other 8 A. S. Narayanan and R. A. Anwar, Biochem. J. 114, 11 (1%9).
[33]
609
PRIMARY STRUCTURE OF INSOLUBLE ELASTIN
25
20-
o
oO
15-
10
i
0
CPA I
CPB
~
'
10
I
20
30
Fraction Number FIG. 2. Chromatographic separation of elastolytic peptides of elastin on cellulose phosphate.
cross-links can be obtained by further fractionation of CPB (Fig. 2) on Sephadex G-50 and Sephadex G-25. Fractions labeled CPB are pooled, dried under reduced pressure, and dissolved in 3-5 ml of 0.01 M NH4HCO3 buffer, pH 8.8. This solution is applied on a Sephadex G-50 (fine) column (4 × 80 cm) previously equilibrated with 0.01 M NH4HCOa buffer, pH 8.8. The column is then eluted with the same buffer at a flow
610
ELASTIN
[33]
1.5
CD
1.0
0.5
0
I
I
i
i
I
20
40
60
80
100
Fraction Number FIG. 3. Chromatography of fraction CPB (Fig. 2) on Sephadex G-50.
rate of 100 ml/hr. Fractions of 10 ml are collected, and their absorbance is measured at 280 nm. A typical elution profile of a Sephadex G-50 chromatography is shown in Fig. 3. The fractions are pooled as indicated in Fig. 3. The pooled fractions appearing under the peak marked G-50-III are dried under reduced pressure on a rotary evaporator and dissolved in about 1 ml of 0.01 M NH4HCO3 buffer, pH 8.8. This solution is applied on to a Sephadex G-25 (fine) column (2.0 × 35 cm), previously equilibrated with 0.01 M NH4HCO3 buffer, pH 8.8, and the column is eluted with the same buffer. Fractions of 5.0 ml are collected, and absorbance is measured at 275 nm after suitable dilution, when necessary. A typical elution profile of Sephadex G-25 chromatography is shown in Fig. 4. The desmosine-containing cross-linked peptides present in fractions labeled G-25.2 are free of other cross-links and contain 25-30% of the total desmosine and isodesmosine present in elastin. Before pooling the fractions, it is prudent to analyze (amino acid analysis) suitable aliquots from individual fractions on either end of the peak marked G-25.2 to ascertain that the fractions are essentially free of cross-links other than the desmosines (see Chapters [16] and [31]).
12C
[33]
PRIMARY STRUCTURE OF INSOLUBLE ELASTIN
611
8.0
6.0
4.0
2.0
G25.1 I
0
•5
15
1'0 Fraction
2'0
Number
FIG. 4. C h r o m a t o g r a p h y of fraction G-50-III (Fig. 3) on Sephadex G-25.
The pooled fractions (G-25.2) are dried under reduced pressure on a rotary evaporator and the dried material is dissolved in 1 ml of pyridinewater-acetic acid ( 4 : 4 : 1) mixture and applied on to a Sephadex LH-20 column (1.9 x 60 cm) previously equilibrated with 50% pyridine solution. The column is eluted with 50% pyridine at a flow rate of 28 ml/hr. Fractions o f 3.5 ml are collected, absorbance is measured at 330 nm, and aliquots (10 /zl) are assayed with ninhydrin after alkaline hydrolysis. Owing to the presence of 50% pyridine in elution solvent, absorbance at 280 nm or 275 nm cannot be measured. The excluded material elutes in fractions labeled A in Fig. 5. These fractions, which contain all the desmosine-containing cross-linked peptides applied to the column, are pooled and dried under reduced pressure on a rotary evaporator. This dried material is used for the release of single-chain peptides from the carboxyl terminals of the desmosine cross-links. As an example of the degree of purification expected at each step, amino acid compositions o f crosslinked bovine elastin peptides at various stages of purification are shown in the table.
25
612
ELASTIN
.....
OL§ V
d I
[33]
o
I
1
0
0
.-~
~
< 0
/
d
0 O
E Z
I
0
0
I
I
I 0
0
d
0
OEE y
0
--
0
[33]
613
PRIMARY STRUCTURE OF INSOLUBLE ELASTIN AMINO ACID COMPOSITION OF CROSS-LINKED BOVINE ELASTIN PEPTIDES AT VARIOUS STAGES OF PURIFICATION a
Amino acid
CPB
G-50-I G-50-II G-50-III G-25.1 G-25.2 G-25.3
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Isodesmosine Desmosine Lysinonorleucine
0.2 0.3 0.6 0.5 4.8 10.4 16.8 1.7 0.6 1.6 0.4 1.2 0.41 0.59 0.1 0.9 1.1
0.4 0.4 0.6 0.7 6.6 15.3 19.6 3.7 0.9 2.7 0.7 2.0 0.42 0.58 0.1 0.4 0.5
Lysine
Arginine
0.1 0.3 0.5 0.5 4.4 14.2 17.6 2.3 0.5 1.8 0.6 1.7 0.35 0.65 0.1 0.3 0.4
0.2 0.3 0.4 0.4 1.5 4.5 11.5 0.8 0.3 0.7 0.3 1.1 0.31 0.69 0.1 0.2 0.3
0.1 0.3 0.4 0.3 2.0 5.7 13.0 0.9 0.2 0.9 0.3 1.0 0.34 0.66 0.1 0.1 0.3
0.1 0.2 0.4 0.3 1.4 4.4 11.4 0.7 0.2 0.7 0.3 1.0 0.33 0.67 -0.1 0.3
0.5 0.4 0.9 1.0 3.1 9.2 16.9 1.5 0.7 1.4 0.4 2.9 0.32 0.68
A 0.1 0.2 0.3 0.3 1.6 4.1 11.6 0.6 0.2 0.7 0.3 1.0 0.32 0.68
1.4
--
0.3 1.4
0.1 0.1
Values are expressed as residues per residue of cross-link (i.e., desmosine plus isodesmosine).
Release of Carboxyl-Terminal Peptides and Separation of the Released Single-Chain Peptides from the Cross-linked Ones. T h e dried m a t e r i a l ( 2 0 - 3 0 ~ m o l o f d e s m o s i n e plus i s o d e s m o s i n e ) is dissolved in 4 ml o f c o u p l i n g buffer (50% p y r i d i n e , 2% t r i e t h y l a m i n e ) in a n a c i d - w a s h e d P y r e x test t u b e , a n d the t u b e a n d the c o n t e n t s are flushed w i t h n i t r o g e n . A t least 50-fold e x c e s s o f p h e n y l i s o t h i o c y a n a t e is a d d e d to the s o l u t i o n a n d the m i x t u r e is i n c u b a t e d u n d e r n i t r o g e n at 50° for 45 rain. T h e r e a c t i o n mixture is t h e n dried on a r o t a r y E v a p o - M i x at 60 ° u n d e r r e d u c e d p r e s s u r e . A f t e r d r y i n g , the v a c u u m is r e l e a s e d u n d e r nitrogen. T h i s is a c c o m p l i s h e d with the u s e o f a t h r e e - w a y s t o p c o c k ; o n e e n d leads to the test t u b e , t h r o u g h r o t a r y E v a p o - M i x c o n n e c t i o n s ; the o t h e r e n d to a v a c u u m p u m p ; a n d the third end to a n i t r o g e n t a n k . T h e dried r e a c t i o n m i x t u r e is i n c u b a t e d w i t h 1 ml o f trifluoroacetic acid at 50 ° for 15 m i n , a n d t r i f l u o r o a c e t i c acid is r e m o v e d u n d e r r e d u c e d p r e s s u r e . T h e r e s i d u e is dissolved in 1.0 ml o f p y r i d i n e - w a t e r - a c e t i c acid (4 : 4 : 1) m i x t u r e a n d applied to a S e p h a d e x L H - 2 0 c o l u m n (1.9 x 60 cm) p r e v i o u s l y e q u i l i b r a t e d with 50% p y r i d i n e . T h e c o l u m n is e l u t e d with 50% p y r i d i n e at a flow rate o f 28 ml/hr. F r a c t i o n s o f 3.5 ml are c o l l e c t e d , a b s o r b a n c e is m e a s u r e d at 330 n m , a n d aliquots (10/zl) are a s s a y e d with n i n h y d r i n after alkaline h y d r o l y s i s . T h e c r o s s - l i n k e d p e p t i d e s elute in
614
ELASTIN
[33]
fractions labeled A, and the released single-chain peptides appear in fractions labeled B (Fig. 5). Fractions under A are pooled and dried, and the Edman degradation and Sephadex LH-20 chromatography are repeated. It may be emphasized that after each cycle of Edman degradation the released single-chain peptides (fraction B, Fig. 5) are separated from the cross-linked peptides (fraction A). Five cycles of Edman degradation can be carried out for this purpose without any problem. It may be mentioned that most of the information becomes available after three cycles of degradation. Purification of Released Single-Chain Peptides. The released singlechain peptides obtained after each cycle of Edman degradation may be purified separately and then sequenced, or these peptides from several cycles may be pooled and then purified. The pooling of released peptides from several cycles allows better recovery of low-yield peptides. Several methods of peptide purification have been described2 '~° In author's laboratory 2'3 initial separation is carried out on a column (0.9 × 55 cm) of Beckman AA-15 resin (a substitute for AA-15 resin is W-2 resin) with pyridine acetate buffers (I: 0.05 N pyridine acetate, pH 2.6; II: 0.10 N pyridine acetate, pH 3.15; III: 0.5 N pyridine acetate, pH 3.7; IV: 2.0 M pyridine acetate, pH 5.0). The column is first developed with a gradient consisting of 300 ml of each of buffers I, II, and III connected in sequence in the order of increasing pH and molarity. This is followed by a gradient consisting of 150 ml of buffers III and IV. The column is eluted at 55° and a flow rate of 42 ml/hr. The peptide peaks are detected with ninhydrin after alkaline hydrolysis. Further purification of peptides (when required) is achieved by paper chromatography (solvent: n-butanol-n-butyl acetate-acetic acid-water, 135 : 6 : 30 : 50) and/or high-voltage paper electrophoresis, in 0.25 M pyridine acetate buffer, pH 3.5. Sequencing of Purified Released Peptides. Methods for sequence analysis of purified single chain peptides have been described. H'12 In the author's laboratory, the purified single-chain peptides, after amino acid analysis, are sequenced manually with the use of Edman degradation followed by dansylation, la It may be emphasized that coupling and cyclization are carried out under nitrogen. Dansyl amino acids are identified by chromatography on polyamide sheets. TM Other elastolytic, cross-linked peptides of elastin (G-50-I and G-50-II) may be examined by a procedure identical to that described above for 9 This series, Vol. 25, Section ~0 This series, Vol. 47, Section 11 This series, VoL 25, Section lz This series, Vol. 47, Section 1.~ W. R. Gray, this series, Vol. ~4 W. R. Gray, this series, Vol.
VI. VII. VII. VIII. 25, p. 333. 25, p. 135.
[34]
B I O S Y N T H E S I S OF I N S O L U B L E E L A S T I N
615
fraction G-25.27 The above approach has been successfully used in a comparative study of elastins from different species and tissues. 2 Simultaneous Sequencing of Two Cross-linked Peptide Chains. Foster and co-workers 4"~ have studied desmosine-containing cross-linked peptides isolated from subtilisin and thermolysin digests of elastin. Peptides were purified by chromatography on ion-exchange resin (Technicon peptide resin, 4% cross-linked, or Aminex 50W-X4, or type-P chromobead) with pyridine acetate buffers and by gel filtration (e.g., Sephadex G-50 or Sephadex G-25). In this approach, cross-linked peptide, judged to be homogeneous, is sequenced on a Beckman sequencer. As two or more chains are sequenced simultaneously, the cross-link positions are assigned on the basis of amino acid residue yield obtained at each cycle of degradation. For example, the sequence data (first cycle, Ala, 127 nmol; second cycle, Ala, 85 nmol; third cycle, Ala 61 nmol; fourth cycle, Ala, 81 nmol; fifth cycle, Ala, 34 nmol) are interpreted to show the sequencing of two chains. At first cycle, both chains contain Ala. At second cycle the yield of Ala is about half that of the first cycle; therefore, one chain has Ala at this position, and in the other chain this position is occupied by the crosslink, and the same is true of the third cycle. At the fourth cycle both positions are assigned to Ala, and in the fifth cycle one position is assigned to Ala and the other to cross-link. As the peptides carboxyl terminal to the cross-links are released, the yield drops sharply and the assignments become difficult. Therefore, in a way this approach is complementary to the one described above. It is apparent that the cross-linked peptide under examination must be essentially homogeneous. It may also be pointed out that amino-terminal analysis alone is not a sufficient proof of homogeneity of desmosine crosslinked peptides, because all desmosine cross-links have alanine residues at the amino terminals and thus will show two alanine residues per residue of desmosine (i.e., desmosine plus isodesmosine) whether the peptide is pure or is a mixture of several peptides.
[34] B i o s y n t h e s i s o f I n s o l u b l e E l a s t i n in C e l l a n d Organ Cultures
By
CARL
FRANZBLAU
and
BARBARA
FARIS
The formation of insoluble elastin in in v#ro systems such as cell and organ cultures has been the focus of several laboratories in recent years. As noted in the overall introduction (Chapter [30]) to the chapters on
METHODS IN ENZYMOLOGY, VOL. 82
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181982-5