BIOSYNTHESIS OF COLLAGEN

THE INFLUENCE OF ASCORBIC ACID ON THE PROLINE, HYDROXY- PROLINE, GLYCINE, AND COLLAGEN CONTENT OF

REGENERATING GUINEA PIG SKIN*

BY BERNARD S. GOULD AND J. FREDERICK WOESSNERt

(From the Division of Biochemistry, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts)

(Received for publication, October 5, 1956)

Ascorbic acid-deficient guinea pigs are unable to repair tissue properly because of their inability to form new collagen. Upon the administration of ascorbic acid this defect is rapidly repaired (1). Among the current hypotheses as to the mechanism involved are suggestions that the defect in scurvy is associated with the inability to produce the precursors of col- lagen or the inability to fibrillate a non-fibrous high molecular weight pre- cursor of almost similar composition to collagen.

Orekhovich and his coworkers (2-4) were able to extract from skin small amounts of an acid-soluble protein, the composition of which resembled collagen. Since the so called “procollagen” was more abundant in young animals than in old, and since much less was found to be present in the skin of scorbutic guinea pigs as compared with that of normal animals, it was assumed to be a precursor. Highberger, Gross, and Schmitt (5) have identified an alkali-soluble collagenous material which has been called tropocollagen. Both of these collagen type fractions have high contents of hydroxyproline, proline, and glycine. Harkness, Marko, Muir, and Neuberger (6) have shown that the alkali-soluble fraction incorporates isotopically labeled glycine relatively rapidly compared to the acid-soluble fraction, making the role of the latter as the major precursor quite improb- able. It is supposed (7) that the protofibrils of collagen consist of an ad- lineation of tropocollagen particles synthesized by the connective tissue cells and aggregated into fibrils in extracellular space.

Earlier studies of collagen biosynthesis by Stetten and Schoenheimer (8) indicated that labeled proline is incorporated into collagen in the form of proline and particularly hydroxyproline. Stetten (9) further showed that labeled hydroxyproline was not incorporated when fed and concluded that the hydroxyproline of collagen is derived from proline. It was suggested

*This investigation was supported by a generous grant from Eli Lilly and Company, to whom we express our thanks, and in part by grant No. A-1270 from the Division of Research Grants and Fellowships, National Institutes of Health, Public Health Service.

t Present address, Biochemistry Laboratories, Howard Hughes Medical Institute, Miami, Florida.

289

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290 BIOSYNTHESIS OF COLLAGEN

that the proline is converted to hydroxyproline after it has been incorpo- rated into a peptide or larger molecule.

It is not improbable that under conditions of ascorbic acid deficiency there may be an accumulation of a more primitive precursor than procol- lagen or tropocollagen, rich in proline and glycine but free of hydroxypro- line. The administration of ascorbic acid might be expected to bring about the hydroxylation of the proline.

Studies of the changes in proline, hydroxyproline, and glycine distribu- tion of granulation tissue in regenerating skin of scorbutic guinea pigs, before and after administration of ascorbic acid, have been undertaken to ascertain whether there is an apparent accumulation of such a precursor which disappears after ascorbic acid administration.

Quantitative studies, to be reported separately, of collagen formation by chick fibroblasts grown in roller tubes in media deficient in or completely lacking ascorbic acid have been carried out in order to learn whether as- corbic acid plays a direct or indirect role in collagen synthesis. Fibroblast growth and collagen formation have been found to proceed normally in media lacking ascorbic acid. Ascorbic acid in combination with whole embryo extract, dialyzed media, or a synthetiti mixture of salts, amino acids, growth factors, and glucose was found to have no significant effect on cell growth or collagen formation. This is in marked contrast to the almost total failure of collagen formation in scorbutic animals and suggests an indirect role for ascorbic acid in the collagen-producing system.

Materials and Methods

Animals---The methods used by Abercrombie et al. (10) for the study of collagen formation in wound healing in rats have been somewhat modified and applied to the study of collagen formation in normal, scorbutic, and ascorbic acid-treated guinea pigs.

Male guinea pigs weighing 250 to 300 gm. and averaging about 275 gm. were placed on a scorbutigenic diet (11) which resulted in signs of scurvy within 13 to 15 days, and death generally occurred in 30 to 34 days. Nor- mal control animals were fed this diet supplemented daily with 20 mg. of n-ascorbic acid given by dropper. After suitable periods, as required by the experiment, the animals were prepared as follows under light ether anesthesia : The hair of the intrascapular region was removed and circles of skin, I.6 cm. in diameter, were removed on both sides of the mid-line. This exposed a fatty pad of tissue beneath the scapulae with several small blood vessels on its surface. These vessels were severed to promote good clot formation. The edge of the cut area was marked carefully with India ink to facilitate later collection of newly formed tissue.

Co&&n of !Pissue---At various intervals after removal of skin, during which time repair had proceeded in so far as possible, certain of the animals

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B. S. GOULD AND J. F. WOESSNER 291

were killed and treated in the following manner. The skin was dissected from the region containing the newly formed tissue. The scab, if present, was discarded and the newly formed connective tissue within the original ink-marked area was carefully dissected, care being taken to exclude pre- existing collagen. The granulation tissue collected in this manner was finely minced in acetone. In many cases, one area was dissected out under light ether anesthesia and the animal replaced on a supplemented diet. After the desired time, the granulation tissue of the second area was col- lected as previously described.

The finely minced tissue was collected by centrifugation or by decanting off the first acetone wash, and then resuspended in acetone for a further 24 hours. The acetone-dried tissue was then defatted with anhydrous ethyl ether. The tissue was transferred to a 13 X 100 mm. tared Pyrex tube, dried overnight at 108” in a vacuum oven, and weighed.

Chemical Methods; Isolation of Collagen-The collagen of the sample was converted to soluble gelatin by adding 3 ml. of water to each sample tube and autoclaving for 3 hours at a steam pressure of 25 pounds per sq. in. The tube was then centrifuged and the extract transferred to a second tube. The residue was reextracted with 2 ml. more of water by autoclaving as before. The extracts were combined and evaporated to dryness in a current of air on a steam bath. Analyses of the residues indicated that no significant amount of collagen remains unextracted.

Hydrolysis of Autoclaved Extract-The autoclave-extractable material was hydrolyzed in 6 N HCl in a sealed tube by heating at 150” for 3 hours. The hydrolysate was, in cases in which high levels of salt did not later interfere, neutralized with the theoretical amount of alkali. Where salt was to be avoided, the excess HCl was eliminated by vacuum distillation.

Hydroxyproline DeterminationsThese were carried out by the method of Neuman and Logan (12) as modified by Martin and Axelrod (13). The hydroxyproline value multiplied by 7.46 was taken as a measure of the collagen content. With this means for the determination of collagen, it must be pointed out that this would include fibrous collagen along with any precollagenous substances containing hydroxyproline.

Proline Determinations-These were carried out both by the method of Troll and Lindsley (14), which is based upon the reaction of proline with ninhydrin after the removal of other amino acids by adsorption on Permutit, and by that of Gould and Shwachman (15) based on the reaction between proline and an alkaline nitroprusside-acetaldehyde reagent. Excellent re- sults have been obtained for the proline content of gelatin and several other proteins by this method. Very good agreement has been obtained between the two methods. In most cases, however, the method of Troll and Lindsley was employed.

Determination of Glycine-The method of Alexander et al. (16) based

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292 BIOSYKTHESIS OF COLLAGEN

upon the formation of formaldehyde when glycine is heated with ninhydrin and the subsequent determination of the formaldehyde by reaction with chromotropic acid has been employed.

CalculationsThe hydroxyproline content of collagen has been taken as 13.4 per cent (12). The proline content was found to be 16.75 per cent, or 1.25 X hydroxyproline. The glycine content of collagen has been found to average 26.8 per cent (17) or twice the hydroxyproline. It has been assumed that no protein other than collagen or some of its precursors con- tains hydroxyproline and therefore the tissue content of this amino acid X 7.46 is a measure of “collagen.” This level of hydroxyproline, when multiplied by 1.25, should yield the level of proline and when multiplied by 2 should yield the percentage of glycine combined as collagen in the tissue. Any excess of proline and of glycine over these calculated quan- tities in a sample has been referred to as excess proline and excess glycine or non-collagenous proline and glycine.

Results

Hydroxyproline Formation in Normal and Xcorbutic Guinea Pigs-A group of animals was placed on a scorbutigenic diet for 7 days, at which time skin circles were removed as described. One group of ten animals (Fig. 1, A) was returned to the diet supplemented by 30 mg. of ascorbic acid daily. Another similar group had skin circles removed after 14 days on the pre- paratory diet and then was replaced on the diet supplemented with ascor- bic acid. Curve A, Fig. 1, presents graphically the average values for apparent collagen produced, as measured by the hydroxyproline increase, during the course of regeneration for both groups of “normal” animals since the results were superimposable. Up to the 4th day after removal of the skin, there appears to be little, if any, new hydroxyproline formation, and it is not unlikely that the collagen found at this time represents an infiltration of collagenous material from the periphery of the area. (This is borne out by analyses of the scabs which were found to contain 1.5 per cent collagen. James (18) reported high hydroxyproline values for fresh scabs on relatively small (0.5 cm.) wounds. This may very likely be due to collagen infiltrating from the periphery of the wound since, in the present study, as the wound size increased less collagen could be found in the scab.) Even on the 6th day there was little hydroxyproline formed. During this period histological examination indicates that cellular organization pre- dominates and that very few fibers are present. However, between the 6th and the 8th days there was a rapid production of hydroxyproline which is accompanied by the histological appearance of fibers. Up through 12 days of regeneration there was an increase in total hydroxyproline. At 14 to 15 days, contraction had proceeded to the point where it was practically impossible to collect material from within the original marked area.

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i3. S. GOULD AND J. F. WOESSNEH 293

Curve D (Fig. 1) shows the results of periodic determinations of hydroxy- proline in regenerating areas in a group of ten animals wounded after 7 days and maintained on a scorbutigenic diet. Again data obtained from animals wounded 14 days after being placed on the diet were found to be superimposable. It appears that wounding after withdrawal of ascorbic acid from the diet for 7 days or more results in essentially complete im-

0 C

A

;; 0 t

-1

- ” 4 0 12 16 20

DAYS AFTER WOUNDING

FIG. 1. Hydroxyproline content of the hydrolysates of autoclave-extracted ma- terial from granulation tissue of guinea pig skin wounds made 7 or 14 days after with- drawal of ascorbic acid from the diet. The hydroxyproline values are multiplied by the factor 7.46 to give a measure of apparent collagen. The values plotted refer to the per cent present in the dry granulation tissue taken for analysis. Curve A, animals replaced on the diet supplemented by 30 mg. of ascorbic acid daily; Curve B, animals wounded 7 days after withdrawal of ascorbic acid, maintained on the scor- butigenic diet for 12 days, and then given 50 mg. of ascorbic acid; Curve C, animals treated as animals in Curve B but wounded after 14 days on preparative diet; Curve D, animals maintained on scorbutigenic diet throughout. The arrow indicates the point at which ascorbic acid was restored to the animals.

pairment of hydroxyproline formation. The persistence of the low level of hydroxyproline in scorbutic wounds (Curve D) suggests that preexisting collagen does not disappear, confirming the observations of Elster (19) and of Robertson (20).

Curves B and C (Fig. 1) present the results of administering, 12 days after wounding, 50 mg. of ascorbic acid daily to animals wounded either 7 (Curve B) or 14 days (Curve C) after withdrawal of ascorbic acid from the diet. The animals represented by Curve B, therefore, were on a scorbuti- genie diet for 19 days and those by Curve C for 26 days.

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294 BIOSYNTHESIS OF COLLAGEN

Within 48 hours after ascorbic acid was administered, the hydroxyproline level of the group for Curve B animals had reached that attained by normal animals (Curve A) in 8 days. Those of the group for Curve C lag slightly and require 6 days to produce a comparable amount of hydroxyproline. Correspondingly, as the withdrawal period before wounding is prolonged, the recovery period is lengthened. A group of five animals wounded after 22 days on the scorbutigenic diet and given 50 mg. of ascorbic acid daily

234567 14

DAYS AFTER WITHDRAWAL OF ASCORBIC ACID

FIG. 2. Hydroxyproline content of hydrolysates of autoclave-extracted material from granulation tissue of guinea pig skin wounds made at various times after with- drawal of ascorbic acid from the diet. Determinations were made in each case 12 days after wounding. (Each point is the average of data from six or more animals.) The hydroxyproline values have been multiplied by the factor 7.46 to give a measure of apparent collagen. The values plotted refer to the per cent present in the dry granulation tissue taken for analysis.

from the 31 to 39 days produced new tissue containing but 0.84 per cent hydroxyproline corresponding to the 6.3 per cent “collagen.” These ani- mals had reached what may be referred to as an irreversible state and, in addition to being unable to produce collagen, showed no improvement from their scorbutic condition.

The results indicate that, while in normal animals there is an appreci- able lag in hydroxyproline formation, scorbutic animals show an almost immediate production of hydroxyproline, once ascorbic acid is administered. It would appear that either the scorbutic regenerating area is in a state of cellular organization that merely requires some effect of ascorbic acid for

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B. S. GOULD AND J. F. WOESSNER 295

the synthesis of hydroxyproline or that some non-fibrous precollagenous material has been accumulated which is rapidly fibrillated upon the dietary administration of ascorbic acid.

In other experiments (Fig. 2) in which groups of animals were wounded 0,2,4, and 7 days after withdrawal of ascorbic acid from the diet and gran- ulation tissue collected 12 days after wounding, it was found that con- siderable hydroxyproline was formed when the wounds were made within 4 days after withdrawal of ascorbic acid, even though no further ascorbic acid is administered. This is particularly interesting since it has recently been shown by Burns, Dayton, and Schulenberg (21) that the half life of labeled ascorbic acid administered to guinea pigs is in the order of 2 to 6 days and averages 4 days.

In another experiment, six animals were wounded without having been placed on a preparative ascorbic acid-free diet and were given 10 mg. of n-ascorbic acid daily for the first 4 days, during which time it is known that cellular organization occurs but that no appreciable amount of hydroxy- proline is formed (see Fig. 1, Curve A). Ascorbic acid was withdrawn for the following 8 days, and the granulation tissue of the area was collected. The mean hydroxyproline content was found to be 4.54 (f0.59 standard deviation) per cent based on the dry weight of the tissue corresponding to an apparent collagen content of 33.9 per cent. It appears, therefore, that the administration of ascorbic acid to animals during the period of cellular invasion of the wound area results in stimulation of hydroxyproline pro- duction. It is noteworthy that the level of hydroxyproline formed in the animals wounded less than 4 days after removal of ascorbic acid from the diet, and then receiving no further ascorbic acid, was found to be essentially the same as that produced by depleted animals in 12 days when ascorbic acid is administered throughout the period of regeneration. This is appar- ent if Curve A, Fig. 1, is compared with the curve of Fig. 2.

Amino Acids of Autoclaved Extracts of Normal and Scorbutic Granulation Tissue-If a precursor, which can be easily converted to collagen under proper conditions, actually accumulates, it might be expected to contain high levels of proline and of glycine and should contain little or no hydroxy- proline, since no hydroxyproline accumulates during the healing period in scorbutic wounds. It might be expected that upon the administration of ascorbic acid there would be a decrease in the excess proline, as well as the excess glycine concomitant with the appearance of the hydroxyproline.

In the present investigation, it has been assumed that such a precursor might resemble collagen or gelatin sufficiently to be extractable by auto- claving in the same way that collagen is extracted by conversion to gelatin. Autoclaved extracts of granulation tissue from normal, scorbutic, and as- corbic acid-treated guinea pigs have accordingly been analyzed for proline, hydroxyproline, and glycine. Because of the difficulties encountered in

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296 BIOSYNTHESIS OF COLLAGEN

collecting the entire contents of a regenerating area quantitatively, in al- most all cases results have been expressed as the per cent of the amino acid based on dry weight of tissue recovered. However, in some instances total amounts of the amino acid in the entire regenerating area have been deter- mined.

TABLE I Injluence of Ascorbic Acid on Distribution of Hydroxyproline,

No. days on scar-

1 butigenic diet beform wounding

Proline, and Glyc; ine in Regenerating Skin

‘reatment after wounding

Ascorbic acid for 12 days

Ascorbic acid for 12 days

Ascorbic acid for 12 days

Ascorbic acid for 12 days

Ascorbic acid for 12 days; then 30 mg. for 3 days

Ascorbic acid for 12 days; then 50 mg. for 3 days

-

.-

n

1.

NO. mimals

Hydroxy- proline

w.

0 6

0 3

0 10

30 9

0 13

per cent

*2.87 f0.39

1.64 zko. 10

0.88 fO.12

3.82 f0.36

2.07 fO.ll

0 10 2.20 f0.33

Pdiine

per cent

4.87 f0.49

4.54 zko.47

3.41 f0.20

4.83 f0.40

3.95 f0.55

3.55 f0.35

Glycine

per cent

7.13 f0.91

5.40 zko.05

4.40 f0.29

8.55 fl.1

4.90 f0.52

4.85 zkO.60

-

1 I

.- P

-

Excess xoline

er c&s

1.28

2.49

2.31

0.05

1.36

0.80

: 1

! P

-

EXCfSS glycine

-

:; m cent

1.39

2.12

2.64

0.91

0.76

c, cent

21.3

12.2

6.6

28.5

15.4

0.45 16.4

-

‘Colla- gen”

* All values are expressed as per cent of total dry weight of tissue and the data are presented as the mean f its standard deviation. Excess proline or glycine re- fers to the non-collagenous proline or glycine calculated on the basis that collagen contains 13.4 per cent hydroxyproline, 16.75 per cent proline, and 26.8 per cent gly- tine. The apparent collagen is calculated from the hydroxyproline value by multi- plying by the factor 7.46.

The results of determinations of proline, hydroxyproline, and glycine in the granulation tissue of several groups of animals treated in a variety of ways are given in Table I. Results of other experiments to determine the levels of these amino acids at various intervals after the administration of ascorbic acid are shown in Figs. 3 and 4. From an examination of Fig. 3, it appears that the administration of ascorbic acid resulted in no immediate appreciable decrease in per cent total proline. This is also borne out by the data obtained from other animals wounded after 7 days on the deficient diet and treated as indicated in Table I. The fact that there is not a sharp decrease in the per cent total proline in the wound extract does not imply

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B. S. GOULD AND J. F. WOESSNER 297

that there is not a decrease in the total proline of the entire wound area. In view of the strong contraction that accompanies healing, it is not un-

0 8 12 13 14 15

DAYS AFTER WOUNDING

FIG. 3. Proline and hydroxyproline content of autoclaved extracts of granulation tissue from guinea pigs restored to ascorbic acid (50 mg. per day) 12 days after having been wounded after 7 days on a preparatory scorbutigenic diet. The curve for ex- cess proline is calculated. The values plotted refer to the per cent present in the dry granulation tissue taken for analysis. The points represent values from one or two animals in each case. The arrow indicates the point at which ascorbic acid was restored to the diet.

TABLE II

Total Amino Acid in Whole Wound Area

Hydroxyproline . Excess proline...............

‘I glycine . Total proline..

“ glycine

Dry weight of tissue..

12 days after wounding*

14 days after wounding (30 mg. ascorbic acid

on 12th and 13th days,

3’s 180 192 230 268

fw. 7.6

Y

98

65 89

180 284

mg. 5.3

Difference

+5 -115 - 103

-50 +20

-ggi * Animals wounded after 7 days on deficient diet and maintained for a further 12

days on same diet.

likely to find the per cent total proline actually increased somewhat, as illustrated in Table II. From Fig. 3, it is evident that with the increase in hydroxyproline there was a drop in the excess proline. Similar changes were observed in other groups of animals (Table I). This would appear to be a presumptive indication that proline-containing material from the wound area was converted to “collagen.”

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298 BIOSYNTHESIS OF COLLAGEN

The data presented in Fig. 4 suggest that an analogous phenomenon is involved with respect to glycine where, upon the administration of ascorbic acid to deficient animals, there was a drop in the excess glycine in the first few days. The data in Table I obtained in other experiments emphasize this point. It has been found, however, that the initial drop in excess glycine is frequently followed by an increase in non-collagen glycine as healing proceeds.

TOTAL GLYCINE

12 13 14 15

DAY’S AFTER WOUNDING FIG. 4. Glycine and hydroxyproline content of autoclaved extracts of granulation

tissue of guinea pigs restored to 30 mg. per day of ascorbic acid 12 days after wound- ing and wounded after 7 days on a preparatory scorbutigenic diet. The curve for excess glycine is calculated. The values plotted refer to the per cent present in the dry granulation tissue taken for analysis. The points represent values from one or two animals in each case. The arrow indicates the point at which ascorbic acid was restored to the diet.

Total Proline, Hydroxyproline, and Glycine in Xcorbutic Wounds and in Wounds after Ascorbic Acid Administration-In a few cases the total amino acid per wound was determined in order to prepare a balance of the changes that accompany ascorbic acid administration and collagen formation. One such example is given in Table II.

It is evident that there was a loss in weight of material in the area due to a macroscopically observable contraction of the wound. The amount of hydroxyproline doubled while the per cent hydroxyproline on a dry weight basis tripled. Total proline dropped, while total glycine increased some- what. The non-collagenous proline dropped in an amount twice the hy-

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B. S. GOULD AND J. F. WOESSNER 299

droxyproline increase. If a correction is made for the slight increase in glycine due to synthesis, the change in excess glycine is also twice the in- crease in hydroxyproline.

Were the non-collagenous or excess amino acids present to some extent in the form of a collagen “precursor,” the excess glycine would be expected to drop about twice as much as the hydroxyproline increase, since the ratio of these amino acids in collagen is 2: 1. The excess proline drop should also be about twice the hydroxyproline increase. Slightly more than 1 mole of proline is required for each new mole of hydroxyproline in the collagen molecule, and 1 mole of proline are probably required for the syn- thesis of each new mole of hydroxyproline (9).

The data agree with the postulated requirements. 60 y of hydroxy- proline are accompanied by a 50 y loss in total proline and a loss of 115 y in excess proline. The former might be associated with the synthesis of new hydroxyproline and the remaining 65 y (115 to 50 7) are associated with this hydroxyproline in the new collagen molecule. The total decrease in non-collagen glycine is equal to 103 y plus 20 y of new glycine formed or 123 y.

Nature of Non-Collagenous Proline-Determination of the free proline content of normal and scorbutic wound tissue indicated that there were no significant differences and that less than 2 per cent of the total proline is present as the free amino acid. A separate communication will deal with the fractionation of granulation tissue. However, it can be reported that partial fractionation of the autoclaved extracts by precipitation of non- gelatinous components with trichloroacetic acid, followed by precipitation of gelatin-like material with tannic acid, indicates that practically all of the proline can be recovered in these two fractions, and therefore the non- collagen proline is probably present as a material of high molecular weight.

SUMMARY

Skin regeneration in normal and scorbutic guinea pigs has been studied by quantitative ,determinations of proline, hydroxyproline, and glycine in hydrolyzed autoclaved extracts of the granulation tissue.

Considerable hydroxyproline formation, which has been taken as a meas- ure of collagen formation, occurs in wounds of previously undepleted ani- mals, even though maintained on a scorbutigenic diet. Animals depleted of ascorbic acid for 4 days show considerable impairment, and 7 day deple- tion results in almost complete cessation of hydroxyproline formation. Impaired hydroxyproline formation appears to be one of the earliest mani- festations of the withdrawal of ascorbic acid.

Depleted animals restored to ascorbic acid at the time of wounding pro- duce the bulk of hydroxyproline between the 6th and 8th days after wound-

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300 BIOSYNTHESIS OF COLLAGEN

ing. Similar animals maintained on the scorbutigenic diet produce no hydroxyproline, but when ascorbic acid is administered to such animals 10 to 12 days after wounding, they produce relatively large amounts within 24 to 48 hours.

Rapid hydroxyproline production in such scorbutic animals upon the administration of ascorbic acid may be due to the conversion of an accumu- lated pool of protein material, rich in proline and glycine, to a more imme- diate collagen precursor rich in hydroxyproline, since the appearance of hydroxyproline is accompanied by a concomitant decrease in the non- collagenous proline and glycine of the granulation tissue.

BIBLIOGRAPHY

1. Wolbach, S. B., and Howe, P. R., Arch. Path., 1, 1 (1926). 2. Orekhovich, V. N., Tustanovskii, A. A., Orekhovich, K. D., and Plotnikova,

N. E., Biokhimiya, 13, 55 (1948). 3. Orekhovich, V. N., Abstracts, 2nd International Congress of Biochemistry,

Paris, 106 (1952). 4. Orekhovich, V. N., Resume des communications, 3” Congres International de

Biochimie, Brussels, 50 (1955). 5. Highberger, J. H., Gross, J., and Schmitt, F. O., Proc. Nat. Acad. SC., 37, 286

(1951). 6. Harkness, R. D., Marko, A. M., Muir, H. M., and Neuberger, A., Biochem. J.,

66, 558 (1954). 7. Schmitt, F. O., Gross, J., and Highberger, J. H., Proc. Nut. Acad. SC., 39, 459

(1953). 8. Stetten, M. R., and Schoenheimer, R., J. Biol. Chem., 163, 113 (1944). 9. Stetten, M. R., J. Biol. Chem., 181, 31 (1949).

10. Abercrombie, M., Flint, M. H., and James, D. W., J. Embryol. and Exp. Morphol., 2, 264 (1954).

11. Gould, B. S., and Shwachman, H., Am. J. Physiol., 136, 485 (1942). 12. Neuman, R. E., and Logan, M. A., J. Biol. Chem., 186, 549 (1950). 13. Martin, C. J., and Axelrod, A. E., Proc. Sot. Exp. Biol. and Med., 93, 461 (1953). 14. Troll, W., and Lindsley, J., J. Biol. Chem., 216, 655 (1955). 15. Gould, B. S., and Shwachman, H., Am. J. Dis. Child., 91, 584 (1956). 16. Alexander, B., Landwehr, G., and Seligman, A. M., J. Biol. Chem., 166.51 (1945). 17. Neuberger, A., Symposia Sot. Exp. Biol., 9, 72 (1955). 18. James, D. W., J. Path. and Bact., 69, 33 (1955). 19. Elster, S. K., J. Biol. Chem., 186, 105 (1950). 20. Robertson, W. v. B., J. Biol. Chem., 187, 673 (1950); 196, 403 (1952). 21. Burns, J. J., Dayton, P. G., and Schulenberg, S., J. Biol. Chem., 218, 15 (1956).

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Bernard S. Gould and J. Frederick WoessnerOF REGENERATING GUINEA PIG SKINGLYCINE, AND COLLAGEN CONTENT

THE PROLINE, HYDROXYPROLINE,INFLUENCE OF ASCORBIC ACID ON BIOSYNTHESIS OF COLLAGEN: THE

1957, 226:289-300.J. Biol. Chem. 

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