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From the Department of Surgical Metabolism and Physiology, Division of Surgery, Walter Reed Army Institute
of Research, Walter Reed Army Medical Center, Washington 12, D. C.
It has been known since before 1870 (1) that collagen swells seconds. After the addition of 35 ml of acetic acid of specified
in dilute acetic acid, loses its fibrillar appearance, and eventually concentration to 240 mg of wet tendon, the mixture was allowed
dissolves, in part, to yield a viscous solution. If the temperature to stand in a tightly stoppered Erlenmeyer flask at room tem-
of this solution is raised steadily, its viscosity decreases, at first perature (20”24”) for 24 hours (Experiment l), or for other spec-
slightly, then sharply, a phenomenon which occurs between 13” ified times, centrifuged at 18,000 r.p.m. for 15 minutes, and fil-
989
990 Chromatographic Fractionation of Collagen Vol. 235, No. 4
RESULTS
total amount of collagendissolved,the chromatogramswere es- components became smeared, and at 78 hours no fractionation
sentially identical. occurred.
In all the chromatograms,the cold collagensolutionswerefirst In a number of runs, we carried out the entire procedurester-
heatedto 40” when they were applied to the columns,that tem- ilely up to the chromatography itself. All solutions used in
perature being maintained during the 7-hour runs. A thiid se- theseproceduresgave negative resultswhen cultured for aerobes
riesof experimentswasperformedto determinethe effect, if any, and anaerobeson thioglycolate broth at 37” in room air, and for
of this temperaturealoneon the chromatographicpattern. Ten- fungi on Sabouraud’sbroth at 37” in room air. Thesechromato-
dons were solubilized in 0.1 M acetic acid as before, and gramswere identical to the onesin which no sterile precautions
the solutionkept sterilein a test tube at 40” for 78hours. Chro-
were taken.
matography wasperformedon aliquotsat g-hour intervals in the
usual manner at 40”. The first three patterns were identical; DISCUSSION
during the next 30 hours, there was a slight continuing increase
of the height of the forepeak while the last three fractions re- An accumulatingbody of evidencefrom physical studiesindi-
mained unchanged; after this time the pattern of the last three catesthat dissolvedcollagengives rise to a polydispersesystem
Chrowzatographic Fractionation of Collagen Vol. 235, No. 4
0.50
TABLE II
Residues per 100 amino acids obtained from forefraction by
chromatography of 0.1 N acetic acid solution of
-a25
collagen, followed by acid hydrolysis
0.10
Glutamic acid................. 21.4
Proline 16.7
s
Hydroxyproline 3.9
Glycine....................... 22.5
Valine........................ 1.5
Lysine........................ 4.1
Ar’ginine...................... 3.1
solutions (below 0.25 M) is interesting in that it contains so few tained at lower acid concentrations, consisting of large molecules
amino acids, and that its hydroxyproline content is so low. It with almost the “classical” amount of hydroxyproline.
is difficult to imagine this component as a “contaminant,” since
it has such large amounts of glycine and proline. Elastin, a SUMMARY
likely candidate, has these amino acids in high concentration, but 1. Rat tail tendon collagen has been solubilized at 3” and at
has several other amino acids which are not found in the forepeak, 20°-24” in acetic acid ranging from 0.005 M to 1.0 M, and the
e.g. alanine, leucine, and phenylalanine. Further, elastin has solutions obtained were chromatographed at 40” on carboxy-
very little aspartic acid, serine, or glutamic acid, which are promi- methyl cellulose columns. The critical salt concentrations of the
nent in the forepeak. This fraction may represent an easily buffers used in the gradient elution system were 0.05 M NaCI,
detachable portion of the collagen structure, and probably is 0.05 M NaHzP04 for the lower buffer, and 0.14 M NaCI, 0.05 M
composed of a number of small peptides and amino acids. NaHzP04 for the upper one. A chromatographic run lasted
Three basic questions emerge from these results: (a) by what about 7 hours. At least 4 components were separated from
internal process is the collagen fractionated? (b) What is the each collagen solution in this way.
source of the forepeak obtained from warm acetic acid solutions 2. The fractionation pattern was markedly dependent upon
below 0.25 M; and (c) what is the nature of the process that occurs the acetic acid concentrations of the different rat tail tendon col-
at an acetic acid concentration between 0.25 NI and 0.50 M, that lagen solutions; below 0.25 M, the solution chromatographed at
produces the different and great quantities of forepeaks and that 40” gave rise to a small forepeak which is probably composed of
leads to the disappearance of most of the three last peaks? lower molecular weight peptides, low in hydroxyproline. At the
With respect to the chromatographic separation of the last higher acid concentrations, the major part of the total chro-
three components, we have no evidence for the nature of the matographable material appeared in the forepeaks which as-
17. MA, T. S., AND ZUAZAGA, G., Id Eng. Chem. Anal. Ed., 14, 22. CRUN, E. M. L., AND DOTY, P., Abstracts of papers of the 1Slst
280 (1942). meeting of the American Chemical Society, Washington, 1957,
18. MOORE, S., SPACEMAN, D. H., AND STEIN, W. H., Anal. Chem., p. 77c.
30, 1185 (1958). 23. GALLOP, P. M., SEIFTER, S., AND MEILMAN, E., Nature, 183,
19. ROSEN, H., Arch. Biochem. Biophys., 67, 10 (1957). 1659 (1959).
20. NEUMAN, R. E., AND LOGAN, M. A., J. Biol. Chem., 184, 299 24. WEIR, C. E., AND CARTER, J., J. Research Nat’l. Bur. Stand-
(1950). ads, 44, 599 (1950).
21. LANGHELD, K., Ber. deut. them. Ges., 42, 2360 (1909). 25. BANGA, I., BALO, J., AND SZABO, D., Nature, 174, 788 (1956).
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