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The solubilization of an elastin-like protein from copper-deficient porcine aorta
BIOCHIMICAET BIOPHYSICAACTA
201
BBA 35362 T H E S O L U B I L I Z A T I O N OF AN E L A S T I N - L I K E P R O T E I N FROM COPPERD E F I C I E N T P O R C I N E AORTA
L. B. S A N D B E R G , T. N. H A C K E T T , JR., AND W. H. C A R N E S ~
Departments of Surgery and Pathology, University of Utah, College of Medicine, Salt Lake City, Utah 84 H2 (U.S.A.) (Received D e c e m b e r 23rd, 1968)
SUMMARY
I. A soluble elastin-like protein is present in the aortas of copper-deficient swine. Studies were undertaken to determine the optimal means of selectively solubilizing this protein. 2. Swelling and solubilization studies on copper-deficient and normal, young, porcine aortas over a range of p H values revealed a marked difference in the behavior of the two tissues. Sharp maxima of swelling and solubilization were observed in the copper-deficient tissue at p H 3 and 7. 3. By further studies of the effects of salt at these pH's, it was determined that a relatively pure elastin-like protein can be solubilized from the copper-deficient aorta at p H 3 and low salt concentration. A method of extracting the aorta under these conditions is described.
INTRODUCTION
Recent reports have indicated that there is a soluble elastin-like protein present in the aortas of copper-deficient swine1, 2. This protein is very probably the noncrosslinked precursor of insoluble elastin. Because of its presence in relatively small amounts, and because there are also considerable quantities of other soluble proteins, it seemed desirable to determine the optimal conditions under which this elastin-like protein is solubilized. It also seemed desirable to select conditions under which minimal amounts of other proteins would be extracted simultaneously. Swelling and solubilization studies carried out on the bovine ligamentum nuchae, an elastin-rich structure, have shown that protein solubilization closely parallels the degree of swelling of that tissue 3. I t was therefore decided to carry out a similar study on copper-deficient and normal porcine aortas, as an approach to the problem of optimal and selective solubilization of the elastin-like protein. This * P r e s e n t address: D e p a r t m e n t of Pathology, UCLA, Center for H e a l t h Sciences, Los Angeles, Calif. 9o o24 U.S.A.
Biochim. Biophys. Acta, 181 (1969) 2 o i - 2 o 7
202
L . B . SANDBERG et
al.
paper describes the results of such experiments, as well as studies of the identity oi proteins solubilized with varying p H and ionic strength. MATERIALS AND METHODS
Materials. Aortas were obtained from freshly killed pigs ranging from 6o to 9 ° days of age. Copper-deficient pigs were raised as previously described 4. Several control animals were raised simultaneously by the addition of adequate amounts of copper to the deficient diet. Solutions. Swelling studies were carried out in lightly buffered solutions in both acid and alkaline solutions. The p H range from 2.2 to 8.0 was covered b y 0.02 M phosphate-citric acid buffer 5. The p H range from 8.6 to lO.6 was covered by 0.02 M glycine-NaOH buffer s. The necessary salt molarities were made by adding NaC1 to the buffer solutions. Swelling procedures. Weighed samples (400-800 rag) of flesh aortas were soaked in 5 ° ml of the various buffered solutions in screw top erlenmeyer flasks at 4 ° with agitation. A crystal of thymol was added to each solution. After three days the specimens were removed from solution, carefully blotted on filter paper with frequent turning for 5 rain and reweighed. Solution p H was determined at the time of specimen removal. Representative fresh aortic samples were also dried at IOO° for 24 h to determine water content. The degree of swelling was calculated by the formula utilized in a previous publication 3, in which swelling is expressed as grams of water uptake per gram dry weight of insoluble protein. Determination of solubilization. Solubilized protein in the swelling solutions was determined by the method of LOWRY et al. ~, as well as by total recovery of amino acids from cold 5% trichloroacetic acid precipitated samples, evaluated on the amino acid analyzer. Standards utilized for the determination of LowRY et al.7 were a-elastin 8 and bovine serum albumin. These two proteins gave very similar results. Amino acid analyses. Amino acid analyses of the hydrolyzed trichloroacetic acid-precipitated samples were performed on a Technicon TSM Amino Acid Analyzer using the physiologic columns, Type C-4 chromobeads, and a modified program and buffer system to inlprove resolution. Hydroxyproline contents were determined by the method of WOESSNER 9. Evaluation of proteins solubilized using amino acid analyses data. Three assumptions were made regarding the protein mixtures evaluated on the amino acid analyzer. First, we assumed that only three major components were present in significant amounts: elastin, ground substance proteins, and collagen. Second, we assumed that collagen is the only component containing significant amounts of hydroxyproline. Third, we assumed that soluble elastin contains no significant amount of aspartic acid. The amino acid contributions from collagen, corresponding to the content of hydroxyproline, were calculated using the composition of purified collagen from pig skin 1°. In correcting for collagen, these contributions were subtracted from the amino acid contents in each of the fractions. The remaining aspartic acid was assumed to represent the ground substance proteins. The content of ground substance proteins was calculated on the basis of lO.2 g residue percent aspartate and 6.0 g residue percent valine ~. In correcting for ground substance proteins, these contributions were " L . B . S A N D B E R G A N n A. L . HODGE, unpublished d a t a . Biochim. Biophys. Acta, 181 (1969) 2Ol-2O7
SOLUBLE ELASTIN FROM COPPER-DEFICIENT AORTA
203
160C • ---•Control normal ~Copper
•
deficient
o
o
~& 120£
j
.~ 8O0
~ 4o ~
oo
',,." ' 41o ' Final pH of solution
a'.o
-
'
12.0
Fig. 1. Swelling of copper-deficient and normal, y o u n g porcine thoracic aortas, o,4-o.8-g portions of t h e whole wet tissue were swelled for 3 days in 5 ° m l of cold o.15 M NaC1 solutions buffered at the various pH's.
again subtracted from the observed amino acid contents which had already been corrected for collagen. The valine residues then remaining were assumed to represent the elastin-like fraction on the basis of its reported amino acid composition 1. RESULTS
Water content of aortic tissues. The analyzed mean water content of 19 aortic specimens was 71.36% with a standard deviation of zk 1.48. No differences were noted in means for normal and copper-deficient tissues. Swdling of normal and copper-deficient aortas as a function of pH. Fig. I shows the swelling of copper-deficient and normal aortas in o.15 M buffered solutions in the pH range of 2.2-1o.6. There is a marked difference in the swelling of the two tissues, copper-deficient aorta showing maxima at pH 3 and 7. These maxima are not observed with the normal tissue. The effects of ionic strength at these two pH maxima, as well as the nature of the proteins solubilized from copper-deficient aorta, were therefore studied further (Figs. 2a and 2b). Solubilization curves for normal and copper-deficient tissue were identical in shape to the swelling curves of Fig. I. Therefore, these plots are not shown. Solubilization of proteins as a function of salt concentration. Figs. 2a and 2b demonstrate the solubilization of aortic proteins at various salt concentrations buffered at both pH 3 and 7. The results of these two experiments are markedly dissimilar. At pH 3 (Fig. 2a), there is maximum solubilization at very low ionic strength. This peak drops off rapidly, reaching a plateau at about 0.2 M salt. Above 0.6 M salt, it drops off again. At pH 7 (Fig. 2b), very little protein is solubilized at low ionic strength. The amount of protein solubilized increases with ionic strength, reaching a peak at from 0. 4 to o.6 M salt. Above this molarity, there is a gradual drop in the amount solubilized. The solubilized protein in these experiments was measured both by the method of LOWRY et al. ~ and by recovery of amino acids from 20 samples analyzed on the TSM analyzer. The dotted line plots shown in Figs. 2a and 2b are drawn on the basis of the amino acids recovered by the latter method, since these results seemed more reliable. The method of LOWRY et al/ evaluates all Biochim. Biophys. Acta, 181 (1969) 2Ol-2O 7
204 ~c
L. B. SANDBERG et h
.
.
.
.
"
pH 3
-
9lb
-
• -•TOtal protein o----e Elatin- like ppotein
7
al.
pH 7 - - ~ [oral protein ~Elastin like protein •
•
L
ii
"-_~
x\
g
\\
Q
} o I
L__
0.4 NclCI (M)
I
0.8
I
0.4
0.3
NaCI ( M )
~
,.;
1.2
Fig. 2. Solubilization of proteins from copper-deficient porcine a o r t a as a function of salt concent r a t i o n at (a) p H 3 and (b) p H 7- Total proteins were evaluated b y recoveries of amino acids from h y d r o l y s a t e s of trichloroacetic acid precipitated protein. The c o n t e n t of soluble elastin was calculated b y the m e t h o d of PETRUSKA AND SANDBERG2.
proteinaceous material solubilized, even small peptides which do not concern us here. Qualitative evaluation of protein solubilized as a function of salt concentration. Using the method of PETRUSKAAND SANDBERG2, a qualitative analysis of the proteins solubilized at each of the points shown in Figs. 2a and 2b was made. Table I gives some representative values of hydroxyproline, aspartic acid, and valine obtained from amino acid analysis and used for these computations. Table I also gives the percentages of collagen, ground substance proteins, and elastin, calculated on the basis of these amino acid contents. Soluble elastin is also represented by the percent difference which remains after subtraction of collagen and ground substance proteins. These latter values are all somewhat higher. It is evident by either method of calculation that elastin-like protein comprises a significant portion of the total protein TABLE I SOME
AMINO
ACID
VALUES
AND
THE
CALCULATED
PROTEIN
CONSTITUENTS
FROM
SOLUBILIZATION
DATA
H y d r o x y p r o l i n e , a s p a r t a t e and valine values were selected from the a m i n o acid analysis data of representative p o i n t s in Fig. 2a (pH 3 values) and Fig. 2b (pH 7 values). Percent of the various protein c o n s t i t u e n t s were calculated from these values.
Swelling solution
Amino acid (g residue percent)
Protein (%)
pH
Hyp
A sp
Val
Collagen
Ground Soluble substance elastin protein by valine content
Soluble elastin by difference
1.88 1.44 1.22 0.69 1.38 1.3o
0.92 1'°9 1.16 5.94 3.8o 5.97
12.13 12"93 11.42 8.94 9.33 7.93
16.1o 12.4o IO.5O 5.90 11.8o 11.2o
o.oo 3.60 5.3 ° 53.90 3O.lO 51.2o
83.90 84.00 84.20 4 °.20 58.IO 37.60
Salt conch.
(M)
3 3 3 7 7 7
-0. 4 0.8 -o-4 o.8
Biochim. Biophys. Aeta, 181 (1969) 2Ol-2O7
72.4 ° 76.5 ° 66.90 34.3 ° 44.6o 28.30
SOLUBLE ELASTIN FROM COPPER-DEFICIENT AORTA
205
solubilized at pH 3 (see solid line plot of Fig. 2a). The maximum amount solubilized corresponds to the point of maximal total protein sohibilization (zero salt concentration of Fig. 2a). The amino acid composition of the cold trichloroacetic acid precipitate at this point of the curve in residues per IOOO residues is as follows: aspartate 7.7, hydroxyproline IO, threonine 15, serine 12, glutamate 21, proline IiO, glycine 322, alanine 218, cystine trace, valine IiO, methionine trace, isoleucine 18, leucine 51, tyrosine 16, phenylalanine 30, lysine 47, histidine trace, arginine 13, desmosine and isodesmosine zero. At pH 7 (Fig. 2b), the elastin-like protein forms a much smaller percentage of the total protein solubilized at all points. At 0.4-0. 5 M NaC1, the curve for total protein as well as the curve for elastin-like protein reaches a peak.
DISCUSSION
Swelling and solubilization. The differences in the swelling and solubilization ot copper-deficient, as compared to normal, aorta are felt to be highly significant. These differences are probably due to the absence of intermolecular cross-links in the copperdeficient material, which greatly reduces the cohesive forces between molecules, allowing increased swelling and solubilization. A copper-containing tissue amine oxidase is presumably necessary for the production of these cross-links in both collagen and elastinn, TM. The swelling of normal aorta is very similar to that reported for collagen by BOWES AND KENTENTM. Thus, it is probable that the large amount of collagen present in aorta is responsible for the swelling curve of the normal tissue, the elastin contributing very little to the swelling observed. The poorly cross-linked elastin in copper deficiency, on the other hand, probably is responsible for the major portion of the two maxima observed for this tissue. It may be that collagen contributes somewhat to this swelling since it also might have fewer cross-links than usual, due to the effect of copper deficiency1.. A superficial similarity exists between the swelling curves of fetal bovine ligamentum nuchae s and copper-deficient aorta. However, the amplitudes of the two peaks are reversed. That is, there is greater swelling of the fetal llgamentum nuchae at high pH, while the reverse is true of copper-deficient aorta. The pH maxima are also different for these two materials. The acidic maximum for copperdeficient aorta occurs at pH 3, while that tor fetal ligamentum nuchae is slightly above pH 2. The basic maximum for copper-deficient aorta is a rather wide peak with a crest at pH 7- The fetal ligamentum nuchae showed a rather sharp peak at pH 8.4. It is surmised that the swelling observed in these two tissues is associated with the sohibilization of markedly different proteins. Extensive investigations (L. B. SANDBERG AND A. J . HODGE, unpublished data) have tailed to reveal the presence of any significant amounts of soluble elastin in the extracts ot normal fetal or calf llgamentum nuchae. Proteins solubilized as a function of ionic strength at p H 3 and 7. Of greatest significance in this study is the large amount of elastin-like protein sohibilized at pH 3. In all samples analyzed, this protein accounted for 67-81~/o of the total (Fig. 2a and Table I). At pH 7, this value never exceeded 500/o (with 0.6 M salt, Fig. 2b). Biockim. Biophvs. Acta, 181 (1969) 2Ol-2O7
206
L.B. SANDBERG et al.
Also of importance is the nature and quantity of other proteins solubilized. The chief contaminant solubilized at p H 3 was collagen, while the contaminants solubilized at pH 7 appeared to be chiefly ground substance proteins. The latter seem to have a low isoelectric point (probably pH 4-5), and therefore are not well solubilized at p H 3 (D. W. SMITH,unpublished data). Some consideration should be given to the validity of the special calculations utilized for determination of the amounts of various protein constituents present in the supernatant solutions after swelling. This method only adapts well to analysis of known protein mixtures where there are unique amino acids present in significant concentration in one protein and/or an absence of some of the common amino acids in one or more of the other constituents. These criteria are well fulfilled because collagen is rich in hydroxyproline, and pure elastin appears to have a near or complete absence of aspartate and hydroxyproline. The weakest point of the argument lies in the fact that ground substance proteins are a heterogeneous mixture with an aspartate content varying by I or 2 g percent. Thus, we feel the error of the method is greatest in evaluating these proteins. Because their valine contents are low and that of elastin is the highest of any known protein, there is little reflection of this variability on the calculations of soluble elastin content. Also, in spite of this error, the general shapes of the curves would not be altered ; and thus all of the conclusions drawn from them are valid. The presence, presumably, of a small amount of cellular proteins gives the elastin content by difference (Table I) a higher value than that calculated on the basis of its valine content. The authors feel calculation of elastin by its valine content to be the most accurate estimation of this protein. Extraction procedure. Further experiments have revealed that more proteins can be solubilized from the aortic tissue b y homogenizing it prior to extraction. Our extraction procedure is, therefore, to mince the flesh aorta into small fragments with a sharp scissors or scalpel, freeze and crush it in a stainless steel mortar 15, add buffer, and homogenize further in a Virtis homogenizer or Waring blendor, and then with agitation to extract this tissue homogenate in the cold. A satisfactory buffer, on the basis of the above experiments, has proven to be o.02 M formic acid, extracting for two 24-h periods with IO ml/g wet tissue. The buffer-tissue mixture at the beginning of each extraction period is adjusted to pH 2.8-3.0 with a few drops of o.I M formic acid. In this manner, one thoracic aorta from a 6o-9o-day-old copper-deficient pig yields 5O-lOO mg of the protein in crude form. Presence of the elastin-like protein can be tested by mixing a portion of the supernatant of a centrifuged extract in question with equal quantities of I.O M phosphate buffer at p H 7, warming to room temperature, and observing for the phenomenon of reversible heat precipitation 1. ACKNOWLEDGMENTS This research was supported by American Heart Association Grant 68-7o7 and National Institutes of Health Grants H E 05 069 and H E 12 561. We gratefully acknowledge the skilled technical assistance of Mrs. Ruth Ann Green and Miss Christine Leak. We also wish to thank Mr. D. W. Smith and Dr. N. Weissman for assistance in raising of the copper-deficient pigs. One of the authors (L.B.S.) is an Established Investigator of the American Heart Association. Biochim. Biophys. Acta, 181 (1969) 2Ol-2O7
SOLUBLE ELASTIN FROM COPPER-DEFICIENT AORTA
207
REFERENCES I D. W. SMITH, N. WEISSMAN AND W. H. CARNES, Biochem. Biophys. Res. Commun., 31 (1968) 3o9. 2 J. A. PETRUSKA AND L. B. SANDBERG, Biochem, Biophys. Res. Commun., 33 (1968) 222. 3 D. S. JACKSON, L. n . SANDBERG AND E. G. CLEARY, Biochem. J., 96 (1965) 813. 4 N. WEISSMAN, D. T. REAY, W. F. COULSON AND W. H. CARNES, Lab. Invest., 14 (1965) 372. 5 T. C. MclLVA1NE, J. Biol. Chem., 49 (1921) 183. 6 S. P. L. SGRENSEN, Methods in Enzymology, Vol. I, A c a d e m i c Press, N e w York, 1955, p. 145. 7 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 8 S. M. PARTRIDGE AND H. F. DAVIS, Biochem. J., 61 (1955) 21. 9 J. F. WOESSNER, JR., Arch. Biochem. Biophys., 93 (1961) 44 o. IO J. E. EASTOE, Biochem. J., 61 (1955) 589. i i S. PINNELL, G. R. MARTIN AND $. GEE, Federation Proc., 27 (1968) 2o91. 12 D. W. BIRD, J. E. SAVAGE AND B. L. O'DELL, _Proc. SOC. Exptl. Biol. Med., 123 (1966) 25o. 13 J. H. B o w E s AND R. H. KENTEN, Biochem. J., 45 (1949) 281. 14 W. S. CHOU, J. E. SAVAGE AND B. L. O'DELL, Proc. Soc. Exptl. Biol. Med., 128 (1968) 948. 15 J. B. GRAESER, J. E. GINSBERG, AND E. FRIEDMANN, J. Biol. Chem., lO 4 (1934) 149.
Biochim. Biophys. Acta, 181 (1969) 2 o i - 2 o 7
201
BBA 35362 T H E S O L U B I L I Z A T I O N OF AN E L A S T I N - L I K E P R O T E I N FROM COPPERD E F I C I E N T P O R C I N E AORTA
L. B. S A N D B E R G , T. N. H A C K E T T , JR., AND W. H. C A R N E S ~
Departments of Surgery and Pathology, University of Utah, College of Medicine, Salt Lake City, Utah 84 H2 (U.S.A.) (Received D e c e m b e r 23rd, 1968)
SUMMARY
I. A soluble elastin-like protein is present in the aortas of copper-deficient swine. Studies were undertaken to determine the optimal means of selectively solubilizing this protein. 2. Swelling and solubilization studies on copper-deficient and normal, young, porcine aortas over a range of p H values revealed a marked difference in the behavior of the two tissues. Sharp maxima of swelling and solubilization were observed in the copper-deficient tissue at p H 3 and 7. 3. By further studies of the effects of salt at these pH's, it was determined that a relatively pure elastin-like protein can be solubilized from the copper-deficient aorta at p H 3 and low salt concentration. A method of extracting the aorta under these conditions is described.
INTRODUCTION
Recent reports have indicated that there is a soluble elastin-like protein present in the aortas of copper-deficient swine1, 2. This protein is very probably the noncrosslinked precursor of insoluble elastin. Because of its presence in relatively small amounts, and because there are also considerable quantities of other soluble proteins, it seemed desirable to determine the optimal conditions under which this elastin-like protein is solubilized. It also seemed desirable to select conditions under which minimal amounts of other proteins would be extracted simultaneously. Swelling and solubilization studies carried out on the bovine ligamentum nuchae, an elastin-rich structure, have shown that protein solubilization closely parallels the degree of swelling of that tissue 3. I t was therefore decided to carry out a similar study on copper-deficient and normal porcine aortas, as an approach to the problem of optimal and selective solubilization of the elastin-like protein. This * P r e s e n t address: D e p a r t m e n t of Pathology, UCLA, Center for H e a l t h Sciences, Los Angeles, Calif. 9o o24 U.S.A.
Biochim. Biophys. Acta, 181 (1969) 2 o i - 2 o 7
202
L . B . SANDBERG et
al.
paper describes the results of such experiments, as well as studies of the identity oi proteins solubilized with varying p H and ionic strength. MATERIALS AND METHODS
Materials. Aortas were obtained from freshly killed pigs ranging from 6o to 9 ° days of age. Copper-deficient pigs were raised as previously described 4. Several control animals were raised simultaneously by the addition of adequate amounts of copper to the deficient diet. Solutions. Swelling studies were carried out in lightly buffered solutions in both acid and alkaline solutions. The p H range from 2.2 to 8.0 was covered b y 0.02 M phosphate-citric acid buffer 5. The p H range from 8.6 to lO.6 was covered by 0.02 M glycine-NaOH buffer s. The necessary salt molarities were made by adding NaC1 to the buffer solutions. Swelling procedures. Weighed samples (400-800 rag) of flesh aortas were soaked in 5 ° ml of the various buffered solutions in screw top erlenmeyer flasks at 4 ° with agitation. A crystal of thymol was added to each solution. After three days the specimens were removed from solution, carefully blotted on filter paper with frequent turning for 5 rain and reweighed. Solution p H was determined at the time of specimen removal. Representative fresh aortic samples were also dried at IOO° for 24 h to determine water content. The degree of swelling was calculated by the formula utilized in a previous publication 3, in which swelling is expressed as grams of water uptake per gram dry weight of insoluble protein. Determination of solubilization. Solubilized protein in the swelling solutions was determined by the method of LOWRY et al. ~, as well as by total recovery of amino acids from cold 5% trichloroacetic acid precipitated samples, evaluated on the amino acid analyzer. Standards utilized for the determination of LowRY et al.7 were a-elastin 8 and bovine serum albumin. These two proteins gave very similar results. Amino acid analyses. Amino acid analyses of the hydrolyzed trichloroacetic acid-precipitated samples were performed on a Technicon TSM Amino Acid Analyzer using the physiologic columns, Type C-4 chromobeads, and a modified program and buffer system to inlprove resolution. Hydroxyproline contents were determined by the method of WOESSNER 9. Evaluation of proteins solubilized using amino acid analyses data. Three assumptions were made regarding the protein mixtures evaluated on the amino acid analyzer. First, we assumed that only three major components were present in significant amounts: elastin, ground substance proteins, and collagen. Second, we assumed that collagen is the only component containing significant amounts of hydroxyproline. Third, we assumed that soluble elastin contains no significant amount of aspartic acid. The amino acid contributions from collagen, corresponding to the content of hydroxyproline, were calculated using the composition of purified collagen from pig skin 1°. In correcting for collagen, these contributions were subtracted from the amino acid contents in each of the fractions. The remaining aspartic acid was assumed to represent the ground substance proteins. The content of ground substance proteins was calculated on the basis of lO.2 g residue percent aspartate and 6.0 g residue percent valine ~. In correcting for ground substance proteins, these contributions were " L . B . S A N D B E R G A N n A. L . HODGE, unpublished d a t a . Biochim. Biophys. Acta, 181 (1969) 2Ol-2O7
SOLUBLE ELASTIN FROM COPPER-DEFICIENT AORTA
203
160C • ---•Control normal ~Copper
•
deficient
o
o
~& 120£
j
.~ 8O0
~ 4o ~
oo
',,." ' 41o ' Final pH of solution
a'.o
-
'
12.0
Fig. 1. Swelling of copper-deficient and normal, y o u n g porcine thoracic aortas, o,4-o.8-g portions of t h e whole wet tissue were swelled for 3 days in 5 ° m l of cold o.15 M NaC1 solutions buffered at the various pH's.
again subtracted from the observed amino acid contents which had already been corrected for collagen. The valine residues then remaining were assumed to represent the elastin-like fraction on the basis of its reported amino acid composition 1. RESULTS
Water content of aortic tissues. The analyzed mean water content of 19 aortic specimens was 71.36% with a standard deviation of zk 1.48. No differences were noted in means for normal and copper-deficient tissues. Swdling of normal and copper-deficient aortas as a function of pH. Fig. I shows the swelling of copper-deficient and normal aortas in o.15 M buffered solutions in the pH range of 2.2-1o.6. There is a marked difference in the swelling of the two tissues, copper-deficient aorta showing maxima at pH 3 and 7. These maxima are not observed with the normal tissue. The effects of ionic strength at these two pH maxima, as well as the nature of the proteins solubilized from copper-deficient aorta, were therefore studied further (Figs. 2a and 2b). Solubilization curves for normal and copper-deficient tissue were identical in shape to the swelling curves of Fig. I. Therefore, these plots are not shown. Solubilization of proteins as a function of salt concentration. Figs. 2a and 2b demonstrate the solubilization of aortic proteins at various salt concentrations buffered at both pH 3 and 7. The results of these two experiments are markedly dissimilar. At pH 3 (Fig. 2a), there is maximum solubilization at very low ionic strength. This peak drops off rapidly, reaching a plateau at about 0.2 M salt. Above 0.6 M salt, it drops off again. At pH 7 (Fig. 2b), very little protein is solubilized at low ionic strength. The amount of protein solubilized increases with ionic strength, reaching a peak at from 0. 4 to o.6 M salt. Above this molarity, there is a gradual drop in the amount solubilized. The solubilized protein in these experiments was measured both by the method of LOWRY et al. ~ and by recovery of amino acids from 20 samples analyzed on the TSM analyzer. The dotted line plots shown in Figs. 2a and 2b are drawn on the basis of the amino acids recovered by the latter method, since these results seemed more reliable. The method of LOWRY et al/ evaluates all Biochim. Biophys. Acta, 181 (1969) 2Ol-2O 7
204 ~c
L. B. SANDBERG et h
.
.
.
.
"
pH 3
-
9lb
-
• -•TOtal protein o----e Elatin- like ppotein
7
al.
pH 7 - - ~ [oral protein ~Elastin like protein •
•
L
ii
"-_~
x\
g
\\
Q
} o I
L__
0.4 NclCI (M)
I
0.8
I
0.4
0.3
NaCI ( M )
~
,.;
1.2
Fig. 2. Solubilization of proteins from copper-deficient porcine a o r t a as a function of salt concent r a t i o n at (a) p H 3 and (b) p H 7- Total proteins were evaluated b y recoveries of amino acids from h y d r o l y s a t e s of trichloroacetic acid precipitated protein. The c o n t e n t of soluble elastin was calculated b y the m e t h o d of PETRUSKA AND SANDBERG2.
proteinaceous material solubilized, even small peptides which do not concern us here. Qualitative evaluation of protein solubilized as a function of salt concentration. Using the method of PETRUSKAAND SANDBERG2, a qualitative analysis of the proteins solubilized at each of the points shown in Figs. 2a and 2b was made. Table I gives some representative values of hydroxyproline, aspartic acid, and valine obtained from amino acid analysis and used for these computations. Table I also gives the percentages of collagen, ground substance proteins, and elastin, calculated on the basis of these amino acid contents. Soluble elastin is also represented by the percent difference which remains after subtraction of collagen and ground substance proteins. These latter values are all somewhat higher. It is evident by either method of calculation that elastin-like protein comprises a significant portion of the total protein TABLE I SOME
AMINO
ACID
VALUES
AND
THE
CALCULATED
PROTEIN
CONSTITUENTS
FROM
SOLUBILIZATION
DATA
H y d r o x y p r o l i n e , a s p a r t a t e and valine values were selected from the a m i n o acid analysis data of representative p o i n t s in Fig. 2a (pH 3 values) and Fig. 2b (pH 7 values). Percent of the various protein c o n s t i t u e n t s were calculated from these values.
Swelling solution
Amino acid (g residue percent)
Protein (%)
pH
Hyp
A sp
Val
Collagen
Ground Soluble substance elastin protein by valine content
Soluble elastin by difference
1.88 1.44 1.22 0.69 1.38 1.3o
0.92 1'°9 1.16 5.94 3.8o 5.97
12.13 12"93 11.42 8.94 9.33 7.93
16.1o 12.4o IO.5O 5.90 11.8o 11.2o
o.oo 3.60 5.3 ° 53.90 3O.lO 51.2o
83.90 84.00 84.20 4 °.20 58.IO 37.60
Salt conch.
(M)
3 3 3 7 7 7
-0. 4 0.8 -o-4 o.8
Biochim. Biophys. Aeta, 181 (1969) 2Ol-2O7
72.4 ° 76.5 ° 66.90 34.3 ° 44.6o 28.30
SOLUBLE ELASTIN FROM COPPER-DEFICIENT AORTA
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solubilized at pH 3 (see solid line plot of Fig. 2a). The maximum amount solubilized corresponds to the point of maximal total protein sohibilization (zero salt concentration of Fig. 2a). The amino acid composition of the cold trichloroacetic acid precipitate at this point of the curve in residues per IOOO residues is as follows: aspartate 7.7, hydroxyproline IO, threonine 15, serine 12, glutamate 21, proline IiO, glycine 322, alanine 218, cystine trace, valine IiO, methionine trace, isoleucine 18, leucine 51, tyrosine 16, phenylalanine 30, lysine 47, histidine trace, arginine 13, desmosine and isodesmosine zero. At pH 7 (Fig. 2b), the elastin-like protein forms a much smaller percentage of the total protein solubilized at all points. At 0.4-0. 5 M NaC1, the curve for total protein as well as the curve for elastin-like protein reaches a peak.
DISCUSSION
Swelling and solubilization. The differences in the swelling and solubilization ot copper-deficient, as compared to normal, aorta are felt to be highly significant. These differences are probably due to the absence of intermolecular cross-links in the copperdeficient material, which greatly reduces the cohesive forces between molecules, allowing increased swelling and solubilization. A copper-containing tissue amine oxidase is presumably necessary for the production of these cross-links in both collagen and elastinn, TM. The swelling of normal aorta is very similar to that reported for collagen by BOWES AND KENTENTM. Thus, it is probable that the large amount of collagen present in aorta is responsible for the swelling curve of the normal tissue, the elastin contributing very little to the swelling observed. The poorly cross-linked elastin in copper deficiency, on the other hand, probably is responsible for the major portion of the two maxima observed for this tissue. It may be that collagen contributes somewhat to this swelling since it also might have fewer cross-links than usual, due to the effect of copper deficiency1.. A superficial similarity exists between the swelling curves of fetal bovine ligamentum nuchae s and copper-deficient aorta. However, the amplitudes of the two peaks are reversed. That is, there is greater swelling of the fetal llgamentum nuchae at high pH, while the reverse is true of copper-deficient aorta. The pH maxima are also different for these two materials. The acidic maximum for copperdeficient aorta occurs at pH 3, while that tor fetal ligamentum nuchae is slightly above pH 2. The basic maximum for copper-deficient aorta is a rather wide peak with a crest at pH 7- The fetal ligamentum nuchae showed a rather sharp peak at pH 8.4. It is surmised that the swelling observed in these two tissues is associated with the sohibilization of markedly different proteins. Extensive investigations (L. B. SANDBERG AND A. J . HODGE, unpublished data) have tailed to reveal the presence of any significant amounts of soluble elastin in the extracts ot normal fetal or calf llgamentum nuchae. Proteins solubilized as a function of ionic strength at p H 3 and 7. Of greatest significance in this study is the large amount of elastin-like protein sohibilized at pH 3. In all samples analyzed, this protein accounted for 67-81~/o of the total (Fig. 2a and Table I). At pH 7, this value never exceeded 500/o (with 0.6 M salt, Fig. 2b). Biockim. Biophvs. Acta, 181 (1969) 2Ol-2O7
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Also of importance is the nature and quantity of other proteins solubilized. The chief contaminant solubilized at p H 3 was collagen, while the contaminants solubilized at pH 7 appeared to be chiefly ground substance proteins. The latter seem to have a low isoelectric point (probably pH 4-5), and therefore are not well solubilized at p H 3 (D. W. SMITH,unpublished data). Some consideration should be given to the validity of the special calculations utilized for determination of the amounts of various protein constituents present in the supernatant solutions after swelling. This method only adapts well to analysis of known protein mixtures where there are unique amino acids present in significant concentration in one protein and/or an absence of some of the common amino acids in one or more of the other constituents. These criteria are well fulfilled because collagen is rich in hydroxyproline, and pure elastin appears to have a near or complete absence of aspartate and hydroxyproline. The weakest point of the argument lies in the fact that ground substance proteins are a heterogeneous mixture with an aspartate content varying by I or 2 g percent. Thus, we feel the error of the method is greatest in evaluating these proteins. Because their valine contents are low and that of elastin is the highest of any known protein, there is little reflection of this variability on the calculations of soluble elastin content. Also, in spite of this error, the general shapes of the curves would not be altered ; and thus all of the conclusions drawn from them are valid. The presence, presumably, of a small amount of cellular proteins gives the elastin content by difference (Table I) a higher value than that calculated on the basis of its valine content. The authors feel calculation of elastin by its valine content to be the most accurate estimation of this protein. Extraction procedure. Further experiments have revealed that more proteins can be solubilized from the aortic tissue b y homogenizing it prior to extraction. Our extraction procedure is, therefore, to mince the flesh aorta into small fragments with a sharp scissors or scalpel, freeze and crush it in a stainless steel mortar 15, add buffer, and homogenize further in a Virtis homogenizer or Waring blendor, and then with agitation to extract this tissue homogenate in the cold. A satisfactory buffer, on the basis of the above experiments, has proven to be o.02 M formic acid, extracting for two 24-h periods with IO ml/g wet tissue. The buffer-tissue mixture at the beginning of each extraction period is adjusted to pH 2.8-3.0 with a few drops of o.I M formic acid. In this manner, one thoracic aorta from a 6o-9o-day-old copper-deficient pig yields 5O-lOO mg of the protein in crude form. Presence of the elastin-like protein can be tested by mixing a portion of the supernatant of a centrifuged extract in question with equal quantities of I.O M phosphate buffer at p H 7, warming to room temperature, and observing for the phenomenon of reversible heat precipitation 1. ACKNOWLEDGMENTS This research was supported by American Heart Association Grant 68-7o7 and National Institutes of Health Grants H E 05 069 and H E 12 561. We gratefully acknowledge the skilled technical assistance of Mrs. Ruth Ann Green and Miss Christine Leak. We also wish to thank Mr. D. W. Smith and Dr. N. Weissman for assistance in raising of the copper-deficient pigs. One of the authors (L.B.S.) is an Established Investigator of the American Heart Association. Biochim. Biophys. Acta, 181 (1969) 2Ol-2O7
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