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A sensitive method for the separation and quantitation of [14C]proline and [14C]hydroxyproline from the collagen of rat uterus
ANALYTICAL
BIOCHEMISTRY
A Sensitive of
61, 513-521 (1974)
Method
for
the
Separation
Quantitation
[ 14C] Proline and [ 14C] Hydroxyproline from the Collagen of Rat Uterus R. A. SALVADOR
Research
and
Division,
Hoffmann-La
Received November
Roche
AND
I. TSAI
Inc., Nutley,
New
Jersey
07110
7, 1973; accepted May 1, 1974
A specific and sensitive method is described for the isolation and quantitation of [Wlproline and Wlhydroxyproline from uterine collagen of the immature rat. Selectivity is achieved in this isolation by using a proteasefree bacterial collagenase. There is complete release of hydroxyproline from uterine protein if the latter is suspended by sonication prior to treatment wit.h collagenase. There is a consistent recovery of C’4CIproline and [%lhydroxyproline when they are added to protein hydrolysates of uterus and then subjected to the procedures required for their isolation and quantitation. It is possible using this method to determine the incorporation of [“Clproline into collagen of the rat uterus and to quantitate its conversion to [“Clhydroxyproline. Coupled with the calorimetric methods for proline and hydroxyproline, it is also possible to determine their specific activity.
It is possible with the use of bacterial collagenase to separate collagenderived peptides from the noncollagenous protein of chick embryo (1,2), bone (3)) and cult,ured fibroblasts (4,5). The use of this method provides the means to examine various metabolic parameters related to alterations in collagen metabolism. An effort was made to apply this procedure to t.he quantitation of the collagen of rat uterus. The difficulties encountered led to the development of a sensitive method for the separation and quantitation of [“Cl proline and [‘“Cl hydroxyproline content of the collagen of this tissue. MATERIALS
AND METHODS
The collagenase used in these experiments was Collagenase ABC, Form III, obtained from Advance Biofactures Corporation, Lynbrook, New Y0rk.l This preparation is devoid of nonspecific proteolytic activity (6). 1 The specific activity of Collagenase ABC, Form III, is 50 units/l0 ~1. A unit is defined by the manufacturer as the amount of collagenase required to solubilize 1 pmole of amino acids (expressed as leucine equiv) from undenatured collagen incubated 24 hr at 37°C. 513 Copyright, @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
514
SALVADOR
AND
TSAI
Both n-[UJ4C]proline and Aquasol were purchased from New England Nuclear while DL- [benzene ring-U-W] tryptophan was obtained from Amersham Searle. N-ethylmaleimide (NEM) was purchased from Sigma Chemical while the cellulose (Avicel, microcrystalline) used to coat thin-layer plates was obtained from Brinkmann Instruments. Immature, female rats of the Long-Evans strain were purchased from Blue Spruce Farms Inc., Altamont, New York. Isolation of total protein from the rat uterus. Immature rats (20 days old) were given an ip injection of either 5 fig estradiol-17/3 or an equal volume of vehicle, daily, for 2 consecutive days. The vehicle consisted of 5% ethyl alcohol in saline. The uterus (cervix and horns) was excised 24 hr after the second dose and weighed. Two uteri were pooled and homogenized in 5% trichloroacetic acid to form a protein pellet. Each protein pellet was washed, dried, and weighed as described previously (7). Some animals were administered 10 ,&i of [14C]proline, iv, 24 hr after either the second dose of saline or estradiol-17/3 and the uteri were removed 4 hr later. Other rats in the estradiol-17P group were administered 5 PCi [14C]tryptophan iv and the uteri were removed 4 hr later. The uteri from rats administered the same radioactive compound were pooled in groups of two and total protein pellets were prepared as described above for nonradioactive uteri. Procedure for the use of collagenase. For collagenase treatment, each pellet is subjected to sonication2 for approximately 30 set in 2 or 3 ml of 0.05 M Tris buffer (pH 7.5) so that each milliliter of suspension contains 5-10 mg of protein. The needle probe is used with a setting of about 45. The volume of sonicate used for collagenase treatment will depend upon the collagen content and/or the amount of radioactivity incorporated into the protein. Sonication is done with the sample in ice to avoid warming. The sonication procedure is repeated from two to six times until a fine suspension is obtained. It is convenient for later steps in the procedure to keep the sonication volume as low as possible. It is important to use no more than 10 mg of protein in each sample since a larger amount of protein interferes with the subsequent separation of [‘“Cl proline and [‘“Cl hydroxyproline by thin-layer chromatography. In the experiments reported here, protein from estradiol-17P and saline treated rats were sonicated in 3 and 2 ml buffer, respectively, and of this, subsequent incubation of 0.8 ml of the former and 1.2 ml of the latter with collagenase released sufficient collagen and radioactivity to be useful for routine assay. The sonicates containing 6-8 mg of protein were incubated with N-ethylmaleimide (5 X 1W M), CaCl, (2 X 1e2 M) and * The sonicator
is a Biosonik
IV;
purchased from Bronwill
Scientific Co.
[‘“cl
PROLINE
AND
[‘“cl
HYDROXYPROLINE
ISOLATIOK
515
150 units of collagenase in a final volume of approximately l-2 ml. These reaction mixtures were prepared in 5 ml conical tubes and were incubated at 37°C for 2 hr with occasional vigorous mixing. At the end of t.he incubation period, a solution of TCA-tannic acid was added to give a final concentration of 5% TCA and 0.25% tannic acid. After cooling 10 min on ice, the tubes were centrifuged to separate the supernatant containing the soluble collagen peptides from the insoluble, noncollagenous protein. The supernatant was transferred to a screw-cap tube that would be suitable for carrying out an acid hydrolysis. The remaining protein pellet was resuspended in 1.0 ml of a cold TCA(5%)-tannic acid (0.25% ) solution, recentrifuged, and the resulting supernatant was added to the original supernatant. The supernatant fraction was then washed by mixing vigorously with an equal volume of cold n-butylacetate (water saturated) to remove most of the TCA (8). The radioactivity contained in the supernatant was determined on a small volume using Aquasol as the scintillat.ion mixture. The peptides in the remaining supernatant were hydrolyzed by adding an equal volume of concentrated HCl and heating at 110°C for 1618 hr. The radioactivity and hydroxyproline content of the noncollagenous protein can also be determined. A portion of a 1.0 ml suspension can be added to Aquasol to determine radioactivity. In the studies reported here, the object was to determine whether collagenase digestion had been complete. Therefore, the suspension of each noncollagenous protein pellet, was hydrolyzed as described above and the hydrolysate was assayed for hydroxyproline using the calorimetric assay (1,9). QzKzntitation of [‘“Cl proline and [W] hydrozyproline in the hydroZyxed collagen. The supernatant fractions obtained through collagenase treatment and hydrolyzed as described above were treated with Norit., centrifuged, and transferred to fresh screw-cap tubes prior to drying in vucuo over NaOH. The resulting residues were dissolved in 1 ml water and were added to Dowex 50(H+) columns that had a capacity of approximately 1.0 ml. Each column was then washed with 10 ml water before eluting the amino acids with 6.0 ml of 3 N NH,OH. The eluate was evaporated in vacua. Each dry residue was dissolved in 1.0 ml water. Of this, 0.1 ml portions were used to analyze for proline and hydroxyproline by the calorimetric methods. The remaining 0.8 ml was dried and redissolved in 0.1 ml for application in a streak on a chromatography plate coated heavily with cellulose (Avicel, microcrystalline). The solvent system consisted of phenol 35:methanol 50:water 15. The usual Rf of hydroxyproline and proline in this system is 0.44 and 0.68 (10). The final extracts applied for chromatographic separation contain a small amount of residual salts which may alter the Rf of the amino acids
516
SALVADOR
AND
TSAI
slightly. This is important since the nonradioactive standards applied directly to the plate will not give an accurate localization of radioactive proline and hydroxyproline so that these areas can be quantitatively removed for the determination of radioactivity. For this reason, it is necessary to add the nonradioactive standards (200 pg each) to the buffer and cofactors used in the collagenase incubation and to subject these samples to the separation and purification procedure. The spray reagent, which is used only for the visualization of the nonradioactive spots of proline and hydroxyproline, consists of 0.2 g ninhydrin dissolved in a mixture of 70 ml methanol, 25 ml water, and 3 ml glacial acetic acid. The plates should be heated to 80°C for good color development. The [‘“Cl proline and [14C ] hydroxyproline standards are prepared by adding each t.o the collagenase digest of control and estradiol-17P uterine protein. These samples are then subjected to the same separation and purification procedures as that used for the unknown samples and the nonradioactive standards. These standards will now give a true localization of [14C]proline and [‘“Cl hydroxyproline on the thin-layer plates. The standards also provide a way of determining the effect of protein on the recovery of each and a correction can be made for incomplete separation. Calorimetric quantitation of hydroxyproline. Certain modifications were introduced int.0 the calorimetric method for hydroxyproline (9) to increase the sensitivity of this analysis. These include reduction of the reaction mixture to 3.0 ml and increasing the concentration of sodium pyrophosphate buffer (pH 8.0) to 100 mM. The chloramine-T concentration (17 mM) was not changed; the reaction was stopped with 0.3 ml of 1 M sodium thiosulfate. Of a 5.0 ml extraction volume of toluene, 4.5 ml was used for the color reaction. Calorimetric quantitation of proline. The method of Troll and Lindsley was used for the quantit.ation of nonradioactive proline (11). Certain modifications were introduced to increase the sensitivity of the analysis. These included a reduction in the volume used to develop t,he ninhydrin color to 4.0 ml and a reduction in the volume of benzene used for extraction to 3.0 ml. RESULTS
Time course of digestion of [14C]proline labeled collagen of rat uterus by collagenuse. The results in Table 1 show that a maximum amount of
radioactivity is solubilized after incubation of rat protein with collagenase for approximately 90 min and that 34% of the radioactivity in the protein is solubilized by this treatment. Extending the incubation to 150 min does not increase the amount of radioactivity which is solubilized.
Time
Course
Incubation (min) 30 60 90 120 0 Portions taining 2800 Collagenase protein was Each rat was 178 and the
[*4C]PRO~~~~
AND
[WIHYDROXYPROLINE
of Digestion
[%]Proline
TABLE Labeled
Radioactivity .___
time Without
collagenase
0 0 -
1 Collagen
of Rat
Uterus
released-cpm With
517
ISOLATION\'
collagenaye 660 884 944 952
by Collagenasen Percent radioactivity solubilized 24 32 34 34
(0.5 ml) of a sonicate of [%]proline-labeled rat uterine protein (3 mg) concpm were incubated as described in Method? for 30, 60, 90, and 120 min. (100 units) was added to all incubations except two controls. The uterine obtained from immature rats treated with 5 rg estradiol-170 for 2 days. administered 10 &i [“Clproline iv 24 hr after the second dose of estradioluterus was removed 4 hr later.
Since tryptophan is incorporated into noncollagenous protein, but not collagen, it serves as a useful marker to test the specificity of collagenase. The results show that essentially no radioactivity is released from [14C]t,ryptophan labeled uterine protein by collagenase but that approximately onethird of the radioactivity is released if the label is [‘4C]proline. If, however, the [‘“Cl tryptophan labeled uterine protein is extracted using the hot trichloroacetic acid procedure (12)) approximately 10% of the radioactivity is solubilieed. Langner and Newman have shown that hot trichloroacetic acid can cause some degradation of collagen (13). Recovery of [14C]proline and [‘“C] hydrozyproline from the cottagena-se digest of uterine protein. It was expected that there would be some loss of proline and hydroxyproline in the procedures for hydrolysis, desalting, and quantitation of the collagenase digest. To determine the extent of this loss, [W]proline and [“Cl hydroxyproline were added to the collagenase digest and the analysis was then completed. The results are given in Table 2. The results indicate that there is a reasonably consistent recovery of [“Cl proline and [‘“Cl hydroxyproline within control and estradiol-17/3 groups but that there is some variation between these groups. This is probably related to some variation in the running of the samples on thin-layer chromatography and is due in part to applying different amounts of protein. The possibility of contaminating the [“Cl hydroxyproline zone by the [‘“Cl proline increases with the application of increasing amounts of protein. It is possible to correct for this by determining the recovery of the [‘“Cl proline and [‘“Cl hydroxyproline standards. Specificity
of collagenuse digestion
of rat uterine protein.
518
SALVADOR
AND
TSAI
TABLE 2 Recovery of [W]Proline and [WlHydroxyproline Standards from the Collagenase Digest of Uterine Proteina Total cpm [Wlproline added to digest
cpm
percent
Control 1 2 3
1440 1440 720
1212 1071 557
84 74 77 ii
720 1440 360
295 871 161
41 61 45 ii
Estradiol-17fi 1 2 3
1440 1440 2160
982 1026 1564
68 71 72 76
720 360 720
431 211 403
60 59 56 58
Protein digest of uterus
Recovery
Total cpm [“ClRecovery hydroxyproline ~ percent added to digest cm
GPortions of a sonicate of uterine protein from control (8 mg) or e&radio&17@ (5 mg) treated rats were incubated with collagenase (150 units) for 2 hr as described in Methods. Then [Wlproline and [14C]hydroxyproline standards were added to the resulting supernatant digest. The remaining procedures for hydrolysis, desalting, and quantitation of this fraction were completed as described in Methods. The recovery of [14C]proline and [14C]hydroxyproline is calculated from losses due to the various methodological manipulrlr tions; this value is used to correct for losses in unknown samples as in Table 4.
Quantitation of [ 14C]proZine and [‘“Cl hydroxyproline in uterine protein from control and estradiol-17/3-treated rats administered [‘“C]proline. The data in Tables 3 and 4 demonstrate that it is possible to quantitate the [“Cl proline and [“Cl hydroxyproline content of collagen of uterine protein of the immature rat following the in vivo administration of [14C]proline. The data in Table 3 shows the recovery of radioactivity from uterine protein following incubation with collagenase. It is apparent that a sufficient quantity of [14C]proline is incorporated into uterine protein in viva so that the amount of radioactivity released from uterine protein by collagenase treatment is sufficient for quantitating [14CJproline and [‘“Cl hydroxyproline. The insoluble protein remaining in each of these samples after collagenase treatment was assayed for hydroxyproline and was found to contain less than 5% of the total hydroxyproline. The collagenase digests were then subjected to procedures for hydrolysis, desalting, and quantitation of [14C]proline and [‘“Cl hydroxyproline as described in Methods. Proline and hydroxyproline were also quantitated by calorimetric methods. The recovery of radioactivity was complete when calculations were corrected for losses during the analytical
[ 14c]
PROLINE
AND
[‘“cl
HYDROXTPROLINE
TABLE
Collagensse Dire&on
of [WlPrnline
3
Lsheled Prot,ein from the IJterus of t,he Rate Radioactivity
Protein digested with collagenase Uterine protein Control Estradiol-17@ 1 2 3 4
619
ISOLATION
released percent
(mg)
Total cpm
6.5
5550
1800
32
5 0
6290 6290 8650 8650
2750 2250 3250 3125
40
5.0 5.9 5.9
cm
37
a Portions of a sonicate of [Wlproline labeled rat ut,erine protein were incubated for 2 hr with 150 units of collagenase BS described in Methods. The immature rats had been treated with either estradiol-178 (5 rg/day) or vehicle (control) for 2 days prior to administering [Wlproline iv. Thii procedure is described in the legend of Table 1 and in Methods. Samples 1, 2 and 3, 4 are duplicates prepared from two different, samples of uterine protein.
procedure. The complete tabulation of results is presented in Table 4. The results in Table 4 show that. it is possible to quantitate the [Ylproline and [14C]hydroxypro1ine in uterine collagen of controI and estradiol-17p stimulated rats following the administration of [l’C]proline in viva. This data is expressed as the cpm of [Wlproline and [14C]hydroxyTABLE
Quantitation
4
of [W]Proline and [WlHydroxyproline in Collagenase Digests of Protein Obtained from the Uterus of the Immature Rat Administered [WlProline In VivoQ Specific activity clzmjmg Radioactivky
Protein digest Control Estradiol-17@ 1 2 3 4
[W]Pro
pqHyPro
cpm [W]Pro: [W]HyPro
[‘“C]Pro/mg Pro
1222
483
2.53
9794
1407 1164
952 1020
1.48
19667
1729
1384
1.14 1.25
13438 12282
1545
1432
1.08
17.567
[‘“ClHyProjmg HyPro 3002
10439 9349 10007 14948
Pro : HyPro 3.26
1.88 1.44
1.23 1.18
a The quantity of [14C]proline ([W]Pro) and [14C]hydroxyproline ([‘%?]HyPro) was determined in the collagenase digest of rat uterine protein that had been labeled in uivo by the administration of [14C]proline. The total quantity of proline and hydroxyproline in this fraction was determined by calorimetric methods.
520
SALVADOR
AND
TSAI
proline found in uterine collagen per se and as the specific activity of these substances. From this data, it is possible to quantitate the conversion of [ 14C]proline to [“Cl hydroxyproline in collagen. As shown by the [ 14C] proline : [“Cl hydroxyproline ratio and the specific activity data, there is considerably more hydroxylation of proline in uterine collagen of the estradiol-17j3 treated rat than in control animals. DISCUSSION
The slow turnover of collagen in most mammalian tissues presents a technical obstacle to examining collagen metabolism. The uterus of the immature rat is useful since the collagen content of this tissue is low and constant, and a marked and rapid increase in this protein can be induced by the administration of estradiol-17P (7). This treatment results in the general growth of the uterus and there is a significant increase in the activity of prolyl hydroxylase (14). Various analogs of proline such as 3,4-dehydroproline and azetidine-2-carboxylic acid have been found to inhibit the formation of collagen in viva in the uterus of the immature rat administered estradiol-17/3 (15). The development of a method to isolate collagen f:om the uterus stems, in part, from a need to examine the effect of these inhibitors on collagen formation. This procedure, like that reported for chick embryo (1,2) and bone (3)) is based on the use of bacterial collagenase. The substrate for collagenase digestions is prepared by sonicating small amounts of uterine protein in Tris buffer. The protein is prepared by homogenizing uteri in trichloroacetic acid and washing with organic solvents (7). The sonication procedure does not cause the release of radioactivity from uterine protein labeled in viva with [‘“Clproline while dissolving protein in NaOH results in a high nonenzymatic blank. Tris buffer should be used since it does not interfere with the separation of [ W] proline and [“Cl hydroxyproline using thin-layer chromatography. Most of the trichloroacetic acid is removed from the sample by washing with n-butylacetate for the same reason. The use of thin-layer chromatography provides a sensitive method for the quantitation of [14C]proline and [14C]hydroxyproline. During this procedure, it is necessary to run recovery standards as described to determine if a correction should be made for contamination of the [14C]hydroxypro1ine area with [‘“Cl proline. The specificity of the bacterial collagenase was established by determining the release of radioactivity from uterine protein of rats administered either [“Cl tryptophan or [14C]proline. Collagenase released 0.5 and 30%, respectively, of the radioactivity contained in the uterine protein of [14C]tryptophan and [14C]proline treated rats. No hydroxyproline remained in the [14C]proline labeled protein after collagenase
[14C]p~o~1~~
AND
[W]HYDROXYPROLIP~E
ISOLATIOR'
521
treatment suggesting that the release of collagen-derived peptides was complete. When [“Cl tryptophan labeled uterine protein was extracted with hot trichloroacetic acid, approximately 10% of the radioactivity was solubilized. It is possible that the procedure described can be applied to other mammalian tissues besides the uterus but this has not been investigated very thoroughly. To do this, it would be essential to determine the recoveries of [ 14C] proline and [‘“Cl hydroxyproline as described. With uterine protein it is possible to recover quantitatively small amounts of these substances in the presence of large amounts of noncollagenous protein. For example, as little as 206 cpm [%]hydroxyproline has been recovered from a collagenase digest of 5 mg of uterine protein. This method would probably not be useful for tissues in which collagen has become refractory to the action of collagenase (16). RE%TRENCES 1. PETERKOFSKY, B., AND DIEGELMANN, R. F. (1971) Biochemistry 10, 988-994. 2. DIEGELMANN, R. F., AND PETERKOFSRY, B. (1972) Develop. Biol. 28, 443-453. 3. DIEGELMANN, R. F., AND PETERKOFSKY, B. (1972) Proc. Nat. Acad. Sci. USA 69, 892-896. 4. PETERKOFSKY, B. (1972) Arch. Biochem. Biophys. 152, 318-328. 5. BATES, C. J., PRYNNE, C. J., AND LEVENE, C. I. (1972) Biochim. Biophys. Acta 263, 397-405. 6. MILLER, R. L., AND UDENFRIEND, S. (1970) Arch. Biochem. Biophys. 139, 104-113. 7. SALVADOR, R. A., AND TSAI, I. (1973) Biochem. Pharmacol. 22, 37-46. 8. YOUNG, D. A. B. (1964) Biochim. Biophys. Acta 90, 178-179. 9. PETERKOFSKY, B., AND PROCKOP, D. J. (1962) Anal. Biochem. 4, 400-406. 10. CHIARI, D., ROHR, M., AND WIDTMANN, G. (1965) Mikrochim. Acta (I$‘&) Heft 4, 669-673. 11. TROLL, W., AND LINDSLEY, J. (1955) J. Biol. Chem. 215, 6&66Q. 12. FITCH, S. M., HARKNESS, M. L. R., AND HARKNESS, R. D. (19.55) Nature (London) 176, 163. 13. LANGNER, R. O., AND NEWMAN, R. A. (1972) Anal. Biochem. 48, 73-79. 14. SALVADOR, R. A., AND TSAI, I. (1973) Arch. Biochem. Biophys. 154, 58%592. 15. SALVADOR, R. A., AND TSAI, I. Unpublished observations. 16. KOHN, R. R., AND ROLLERSON, E. (1960) J. Gerontol. 15, m-14.
BIOCHEMISTRY
A Sensitive of
61, 513-521 (1974)
Method
for
the
Separation
Quantitation
[ 14C] Proline and [ 14C] Hydroxyproline from the Collagen of Rat Uterus R. A. SALVADOR
Research
and
Division,
Hoffmann-La
Received November
Roche
AND
I. TSAI
Inc., Nutley,
New
Jersey
07110
7, 1973; accepted May 1, 1974
A specific and sensitive method is described for the isolation and quantitation of [Wlproline and Wlhydroxyproline from uterine collagen of the immature rat. Selectivity is achieved in this isolation by using a proteasefree bacterial collagenase. There is complete release of hydroxyproline from uterine protein if the latter is suspended by sonication prior to treatment wit.h collagenase. There is a consistent recovery of C’4CIproline and [%lhydroxyproline when they are added to protein hydrolysates of uterus and then subjected to the procedures required for their isolation and quantitation. It is possible using this method to determine the incorporation of [“Clproline into collagen of the rat uterus and to quantitate its conversion to [“Clhydroxyproline. Coupled with the calorimetric methods for proline and hydroxyproline, it is also possible to determine their specific activity.
It is possible with the use of bacterial collagenase to separate collagenderived peptides from the noncollagenous protein of chick embryo (1,2), bone (3)) and cult,ured fibroblasts (4,5). The use of this method provides the means to examine various metabolic parameters related to alterations in collagen metabolism. An effort was made to apply this procedure to t.he quantitation of the collagen of rat uterus. The difficulties encountered led to the development of a sensitive method for the separation and quantitation of [“Cl proline and [‘“Cl hydroxyproline content of the collagen of this tissue. MATERIALS
AND METHODS
The collagenase used in these experiments was Collagenase ABC, Form III, obtained from Advance Biofactures Corporation, Lynbrook, New Y0rk.l This preparation is devoid of nonspecific proteolytic activity (6). 1 The specific activity of Collagenase ABC, Form III, is 50 units/l0 ~1. A unit is defined by the manufacturer as the amount of collagenase required to solubilize 1 pmole of amino acids (expressed as leucine equiv) from undenatured collagen incubated 24 hr at 37°C. 513 Copyright, @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
514
SALVADOR
AND
TSAI
Both n-[UJ4C]proline and Aquasol were purchased from New England Nuclear while DL- [benzene ring-U-W] tryptophan was obtained from Amersham Searle. N-ethylmaleimide (NEM) was purchased from Sigma Chemical while the cellulose (Avicel, microcrystalline) used to coat thin-layer plates was obtained from Brinkmann Instruments. Immature, female rats of the Long-Evans strain were purchased from Blue Spruce Farms Inc., Altamont, New York. Isolation of total protein from the rat uterus. Immature rats (20 days old) were given an ip injection of either 5 fig estradiol-17/3 or an equal volume of vehicle, daily, for 2 consecutive days. The vehicle consisted of 5% ethyl alcohol in saline. The uterus (cervix and horns) was excised 24 hr after the second dose and weighed. Two uteri were pooled and homogenized in 5% trichloroacetic acid to form a protein pellet. Each protein pellet was washed, dried, and weighed as described previously (7). Some animals were administered 10 ,&i of [14C]proline, iv, 24 hr after either the second dose of saline or estradiol-17/3 and the uteri were removed 4 hr later. Other rats in the estradiol-17P group were administered 5 PCi [14C]tryptophan iv and the uteri were removed 4 hr later. The uteri from rats administered the same radioactive compound were pooled in groups of two and total protein pellets were prepared as described above for nonradioactive uteri. Procedure for the use of collagenase. For collagenase treatment, each pellet is subjected to sonication2 for approximately 30 set in 2 or 3 ml of 0.05 M Tris buffer (pH 7.5) so that each milliliter of suspension contains 5-10 mg of protein. The needle probe is used with a setting of about 45. The volume of sonicate used for collagenase treatment will depend upon the collagen content and/or the amount of radioactivity incorporated into the protein. Sonication is done with the sample in ice to avoid warming. The sonication procedure is repeated from two to six times until a fine suspension is obtained. It is convenient for later steps in the procedure to keep the sonication volume as low as possible. It is important to use no more than 10 mg of protein in each sample since a larger amount of protein interferes with the subsequent separation of [‘“Cl proline and [‘“Cl hydroxyproline by thin-layer chromatography. In the experiments reported here, protein from estradiol-17P and saline treated rats were sonicated in 3 and 2 ml buffer, respectively, and of this, subsequent incubation of 0.8 ml of the former and 1.2 ml of the latter with collagenase released sufficient collagen and radioactivity to be useful for routine assay. The sonicates containing 6-8 mg of protein were incubated with N-ethylmaleimide (5 X 1W M), CaCl, (2 X 1e2 M) and * The sonicator
is a Biosonik
IV;
purchased from Bronwill
Scientific Co.
[‘“cl
PROLINE
AND
[‘“cl
HYDROXYPROLINE
ISOLATIOK
515
150 units of collagenase in a final volume of approximately l-2 ml. These reaction mixtures were prepared in 5 ml conical tubes and were incubated at 37°C for 2 hr with occasional vigorous mixing. At the end of t.he incubation period, a solution of TCA-tannic acid was added to give a final concentration of 5% TCA and 0.25% tannic acid. After cooling 10 min on ice, the tubes were centrifuged to separate the supernatant containing the soluble collagen peptides from the insoluble, noncollagenous protein. The supernatant was transferred to a screw-cap tube that would be suitable for carrying out an acid hydrolysis. The remaining protein pellet was resuspended in 1.0 ml of a cold TCA(5%)-tannic acid (0.25% ) solution, recentrifuged, and the resulting supernatant was added to the original supernatant. The supernatant fraction was then washed by mixing vigorously with an equal volume of cold n-butylacetate (water saturated) to remove most of the TCA (8). The radioactivity contained in the supernatant was determined on a small volume using Aquasol as the scintillat.ion mixture. The peptides in the remaining supernatant were hydrolyzed by adding an equal volume of concentrated HCl and heating at 110°C for 1618 hr. The radioactivity and hydroxyproline content of the noncollagenous protein can also be determined. A portion of a 1.0 ml suspension can be added to Aquasol to determine radioactivity. In the studies reported here, the object was to determine whether collagenase digestion had been complete. Therefore, the suspension of each noncollagenous protein pellet, was hydrolyzed as described above and the hydrolysate was assayed for hydroxyproline using the calorimetric assay (1,9). QzKzntitation of [‘“Cl proline and [W] hydrozyproline in the hydroZyxed collagen. The supernatant fractions obtained through collagenase treatment and hydrolyzed as described above were treated with Norit., centrifuged, and transferred to fresh screw-cap tubes prior to drying in vucuo over NaOH. The resulting residues were dissolved in 1 ml water and were added to Dowex 50(H+) columns that had a capacity of approximately 1.0 ml. Each column was then washed with 10 ml water before eluting the amino acids with 6.0 ml of 3 N NH,OH. The eluate was evaporated in vacua. Each dry residue was dissolved in 1.0 ml water. Of this, 0.1 ml portions were used to analyze for proline and hydroxyproline by the calorimetric methods. The remaining 0.8 ml was dried and redissolved in 0.1 ml for application in a streak on a chromatography plate coated heavily with cellulose (Avicel, microcrystalline). The solvent system consisted of phenol 35:methanol 50:water 15. The usual Rf of hydroxyproline and proline in this system is 0.44 and 0.68 (10). The final extracts applied for chromatographic separation contain a small amount of residual salts which may alter the Rf of the amino acids
516
SALVADOR
AND
TSAI
slightly. This is important since the nonradioactive standards applied directly to the plate will not give an accurate localization of radioactive proline and hydroxyproline so that these areas can be quantitatively removed for the determination of radioactivity. For this reason, it is necessary to add the nonradioactive standards (200 pg each) to the buffer and cofactors used in the collagenase incubation and to subject these samples to the separation and purification procedure. The spray reagent, which is used only for the visualization of the nonradioactive spots of proline and hydroxyproline, consists of 0.2 g ninhydrin dissolved in a mixture of 70 ml methanol, 25 ml water, and 3 ml glacial acetic acid. The plates should be heated to 80°C for good color development. The [‘“Cl proline and [14C ] hydroxyproline standards are prepared by adding each t.o the collagenase digest of control and estradiol-17P uterine protein. These samples are then subjected to the same separation and purification procedures as that used for the unknown samples and the nonradioactive standards. These standards will now give a true localization of [14C]proline and [‘“Cl hydroxyproline on the thin-layer plates. The standards also provide a way of determining the effect of protein on the recovery of each and a correction can be made for incomplete separation. Calorimetric quantitation of hydroxyproline. Certain modifications were introduced int.0 the calorimetric method for hydroxyproline (9) to increase the sensitivity of this analysis. These include reduction of the reaction mixture to 3.0 ml and increasing the concentration of sodium pyrophosphate buffer (pH 8.0) to 100 mM. The chloramine-T concentration (17 mM) was not changed; the reaction was stopped with 0.3 ml of 1 M sodium thiosulfate. Of a 5.0 ml extraction volume of toluene, 4.5 ml was used for the color reaction. Calorimetric quantitation of proline. The method of Troll and Lindsley was used for the quantit.ation of nonradioactive proline (11). Certain modifications were introduced to increase the sensitivity of the analysis. These included a reduction in the volume used to develop t,he ninhydrin color to 4.0 ml and a reduction in the volume of benzene used for extraction to 3.0 ml. RESULTS
Time course of digestion of [14C]proline labeled collagen of rat uterus by collagenuse. The results in Table 1 show that a maximum amount of
radioactivity is solubilized after incubation of rat protein with collagenase for approximately 90 min and that 34% of the radioactivity in the protein is solubilized by this treatment. Extending the incubation to 150 min does not increase the amount of radioactivity which is solubilized.
Time
Course
Incubation (min) 30 60 90 120 0 Portions taining 2800 Collagenase protein was Each rat was 178 and the
[*4C]PRO~~~~
AND
[WIHYDROXYPROLINE
of Digestion
[%]Proline
TABLE Labeled
Radioactivity .___
time Without
collagenase
0 0 -
1 Collagen
of Rat
Uterus
released-cpm With
517
ISOLATION\'
collagenaye 660 884 944 952
by Collagenasen Percent radioactivity solubilized 24 32 34 34
(0.5 ml) of a sonicate of [%]proline-labeled rat uterine protein (3 mg) concpm were incubated as described in Method? for 30, 60, 90, and 120 min. (100 units) was added to all incubations except two controls. The uterine obtained from immature rats treated with 5 rg estradiol-170 for 2 days. administered 10 &i [“Clproline iv 24 hr after the second dose of estradioluterus was removed 4 hr later.
Since tryptophan is incorporated into noncollagenous protein, but not collagen, it serves as a useful marker to test the specificity of collagenase. The results show that essentially no radioactivity is released from [14C]t,ryptophan labeled uterine protein by collagenase but that approximately onethird of the radioactivity is released if the label is [‘4C]proline. If, however, the [‘“Cl tryptophan labeled uterine protein is extracted using the hot trichloroacetic acid procedure (12)) approximately 10% of the radioactivity is solubilieed. Langner and Newman have shown that hot trichloroacetic acid can cause some degradation of collagen (13). Recovery of [14C]proline and [‘“C] hydrozyproline from the cottagena-se digest of uterine protein. It was expected that there would be some loss of proline and hydroxyproline in the procedures for hydrolysis, desalting, and quantitation of the collagenase digest. To determine the extent of this loss, [W]proline and [“Cl hydroxyproline were added to the collagenase digest and the analysis was then completed. The results are given in Table 2. The results indicate that there is a reasonably consistent recovery of [“Cl proline and [‘“Cl hydroxyproline within control and estradiol-17/3 groups but that there is some variation between these groups. This is probably related to some variation in the running of the samples on thin-layer chromatography and is due in part to applying different amounts of protein. The possibility of contaminating the [“Cl hydroxyproline zone by the [‘“Cl proline increases with the application of increasing amounts of protein. It is possible to correct for this by determining the recovery of the [‘“Cl proline and [‘“Cl hydroxyproline standards. Specificity
of collagenuse digestion
of rat uterine protein.
518
SALVADOR
AND
TSAI
TABLE 2 Recovery of [W]Proline and [WlHydroxyproline Standards from the Collagenase Digest of Uterine Proteina Total cpm [Wlproline added to digest
cpm
percent
Control 1 2 3
1440 1440 720
1212 1071 557
84 74 77 ii
720 1440 360
295 871 161
41 61 45 ii
Estradiol-17fi 1 2 3
1440 1440 2160
982 1026 1564
68 71 72 76
720 360 720
431 211 403
60 59 56 58
Protein digest of uterus
Recovery
Total cpm [“ClRecovery hydroxyproline ~ percent added to digest cm
GPortions of a sonicate of uterine protein from control (8 mg) or e&radio&17@ (5 mg) treated rats were incubated with collagenase (150 units) for 2 hr as described in Methods. Then [Wlproline and [14C]hydroxyproline standards were added to the resulting supernatant digest. The remaining procedures for hydrolysis, desalting, and quantitation of this fraction were completed as described in Methods. The recovery of [14C]proline and [14C]hydroxyproline is calculated from losses due to the various methodological manipulrlr tions; this value is used to correct for losses in unknown samples as in Table 4.
Quantitation of [ 14C]proZine and [‘“Cl hydroxyproline in uterine protein from control and estradiol-17/3-treated rats administered [‘“C]proline. The data in Tables 3 and 4 demonstrate that it is possible to quantitate the [“Cl proline and [“Cl hydroxyproline content of collagen of uterine protein of the immature rat following the in vivo administration of [14C]proline. The data in Table 3 shows the recovery of radioactivity from uterine protein following incubation with collagenase. It is apparent that a sufficient quantity of [14C]proline is incorporated into uterine protein in viva so that the amount of radioactivity released from uterine protein by collagenase treatment is sufficient for quantitating [14CJproline and [‘“Cl hydroxyproline. The insoluble protein remaining in each of these samples after collagenase treatment was assayed for hydroxyproline and was found to contain less than 5% of the total hydroxyproline. The collagenase digests were then subjected to procedures for hydrolysis, desalting, and quantitation of [14C]proline and [‘“Cl hydroxyproline as described in Methods. Proline and hydroxyproline were also quantitated by calorimetric methods. The recovery of radioactivity was complete when calculations were corrected for losses during the analytical
[ 14c]
PROLINE
AND
[‘“cl
HYDROXTPROLINE
TABLE
Collagensse Dire&on
of [WlPrnline
3
Lsheled Prot,ein from the IJterus of t,he Rate Radioactivity
Protein digested with collagenase Uterine protein Control Estradiol-17@ 1 2 3 4
619
ISOLATION
released percent
(mg)
Total cpm
6.5
5550
1800
32
5 0
6290 6290 8650 8650
2750 2250 3250 3125
40
5.0 5.9 5.9
cm
37
a Portions of a sonicate of [Wlproline labeled rat ut,erine protein were incubated for 2 hr with 150 units of collagenase BS described in Methods. The immature rats had been treated with either estradiol-178 (5 rg/day) or vehicle (control) for 2 days prior to administering [Wlproline iv. Thii procedure is described in the legend of Table 1 and in Methods. Samples 1, 2 and 3, 4 are duplicates prepared from two different, samples of uterine protein.
procedure. The complete tabulation of results is presented in Table 4. The results in Table 4 show that. it is possible to quantitate the [Ylproline and [14C]hydroxypro1ine in uterine collagen of controI and estradiol-17p stimulated rats following the administration of [l’C]proline in viva. This data is expressed as the cpm of [Wlproline and [14C]hydroxyTABLE
Quantitation
4
of [W]Proline and [WlHydroxyproline in Collagenase Digests of Protein Obtained from the Uterus of the Immature Rat Administered [WlProline In VivoQ Specific activity clzmjmg Radioactivky
Protein digest Control Estradiol-17@ 1 2 3 4
[W]Pro
pqHyPro
cpm [W]Pro: [W]HyPro
[‘“C]Pro/mg Pro
1222
483
2.53
9794
1407 1164
952 1020
1.48
19667
1729
1384
1.14 1.25
13438 12282
1545
1432
1.08
17.567
[‘“ClHyProjmg HyPro 3002
10439 9349 10007 14948
Pro : HyPro 3.26
1.88 1.44
1.23 1.18
a The quantity of [14C]proline ([W]Pro) and [14C]hydroxyproline ([‘%?]HyPro) was determined in the collagenase digest of rat uterine protein that had been labeled in uivo by the administration of [14C]proline. The total quantity of proline and hydroxyproline in this fraction was determined by calorimetric methods.
520
SALVADOR
AND
TSAI
proline found in uterine collagen per se and as the specific activity of these substances. From this data, it is possible to quantitate the conversion of [ 14C]proline to [“Cl hydroxyproline in collagen. As shown by the [ 14C] proline : [“Cl hydroxyproline ratio and the specific activity data, there is considerably more hydroxylation of proline in uterine collagen of the estradiol-17j3 treated rat than in control animals. DISCUSSION
The slow turnover of collagen in most mammalian tissues presents a technical obstacle to examining collagen metabolism. The uterus of the immature rat is useful since the collagen content of this tissue is low and constant, and a marked and rapid increase in this protein can be induced by the administration of estradiol-17P (7). This treatment results in the general growth of the uterus and there is a significant increase in the activity of prolyl hydroxylase (14). Various analogs of proline such as 3,4-dehydroproline and azetidine-2-carboxylic acid have been found to inhibit the formation of collagen in viva in the uterus of the immature rat administered estradiol-17/3 (15). The development of a method to isolate collagen f:om the uterus stems, in part, from a need to examine the effect of these inhibitors on collagen formation. This procedure, like that reported for chick embryo (1,2) and bone (3)) is based on the use of bacterial collagenase. The substrate for collagenase digestions is prepared by sonicating small amounts of uterine protein in Tris buffer. The protein is prepared by homogenizing uteri in trichloroacetic acid and washing with organic solvents (7). The sonication procedure does not cause the release of radioactivity from uterine protein labeled in viva with [‘“Clproline while dissolving protein in NaOH results in a high nonenzymatic blank. Tris buffer should be used since it does not interfere with the separation of [ W] proline and [“Cl hydroxyproline using thin-layer chromatography. Most of the trichloroacetic acid is removed from the sample by washing with n-butylacetate for the same reason. The use of thin-layer chromatography provides a sensitive method for the quantitation of [14C]proline and [14C]hydroxyproline. During this procedure, it is necessary to run recovery standards as described to determine if a correction should be made for contamination of the [14C]hydroxypro1ine area with [‘“Cl proline. The specificity of the bacterial collagenase was established by determining the release of radioactivity from uterine protein of rats administered either [“Cl tryptophan or [14C]proline. Collagenase released 0.5 and 30%, respectively, of the radioactivity contained in the uterine protein of [14C]tryptophan and [14C]proline treated rats. No hydroxyproline remained in the [14C]proline labeled protein after collagenase
[14C]p~o~1~~
AND
[W]HYDROXYPROLIP~E
ISOLATIOR'
521
treatment suggesting that the release of collagen-derived peptides was complete. When [“Cl tryptophan labeled uterine protein was extracted with hot trichloroacetic acid, approximately 10% of the radioactivity was solubilized. It is possible that the procedure described can be applied to other mammalian tissues besides the uterus but this has not been investigated very thoroughly. To do this, it would be essential to determine the recoveries of [ 14C] proline and [‘“Cl hydroxyproline as described. With uterine protein it is possible to recover quantitatively small amounts of these substances in the presence of large amounts of noncollagenous protein. For example, as little as 206 cpm [%]hydroxyproline has been recovered from a collagenase digest of 5 mg of uterine protein. This method would probably not be useful for tissues in which collagen has become refractory to the action of collagenase (16). RE%TRENCES 1. PETERKOFSKY, B., AND DIEGELMANN, R. F. (1971) Biochemistry 10, 988-994. 2. DIEGELMANN, R. F., AND PETERKOFSRY, B. (1972) Develop. Biol. 28, 443-453. 3. DIEGELMANN, R. F., AND PETERKOFSKY, B. (1972) Proc. Nat. Acad. Sci. USA 69, 892-896. 4. PETERKOFSKY, B. (1972) Arch. Biochem. Biophys. 152, 318-328. 5. BATES, C. J., PRYNNE, C. J., AND LEVENE, C. I. (1972) Biochim. Biophys. Acta 263, 397-405. 6. MILLER, R. L., AND UDENFRIEND, S. (1970) Arch. Biochem. Biophys. 139, 104-113. 7. SALVADOR, R. A., AND TSAI, I. (1973) Biochem. Pharmacol. 22, 37-46. 8. YOUNG, D. A. B. (1964) Biochim. Biophys. Acta 90, 178-179. 9. PETERKOFSKY, B., AND PROCKOP, D. J. (1962) Anal. Biochem. 4, 400-406. 10. CHIARI, D., ROHR, M., AND WIDTMANN, G. (1965) Mikrochim. Acta (I$‘&) Heft 4, 669-673. 11. TROLL, W., AND LINDSLEY, J. (1955) J. Biol. Chem. 215, 6&66Q. 12. FITCH, S. M., HARKNESS, M. L. R., AND HARKNESS, R. D. (19.55) Nature (London) 176, 163. 13. LANGNER, R. O., AND NEWMAN, R. A. (1972) Anal. Biochem. 48, 73-79. 14. SALVADOR, R. A., AND TSAI, I. (1973) Arch. Biochem. Biophys. 154, 58%592. 15. SALVADOR, R. A., AND TSAI, I. Unpublished observations. 16. KOHN, R. R., AND ROLLERSON, E. (1960) J. Gerontol. 15, m-14.