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The effect of collagenase and trypsin on collagen. An electron microscopic study

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T H E E F F E C T O F COLLAGENASE AND TRYPSIN
ON COLLAGEN
A N ELECTRON MICROSCOPIC STUDY
MADELIXE K. KEECH'
Section of PreveiLtive Medicine, Pale University School of Medicine,
N e w Haven, Connecticiit
FOURTEEN F I G W E S
Banfield ('52) has recently drawn attention to the presence
of collagen fibrils with tapered ends in sites where collagen
was apparently being actively formed (embryonic human
tissue and mesenchymal neoplasms). I n preparations of
adult tissue such fibrils were rare or absent. Randall,
Fraser, Jackson, Martin and North ( '52) reported tapered
collagen fibrils from the umbilical cord of the rat and suggested that collagen fiber growth may take place by a process of pyramidal accretion. Noda and Wyckoff' ('51) noted
fibrils terminating at one or both ends as cones tapering to
a point in reprecipitated collagen using citrate buffer a t pH
4.6. Gross ('53) illustrated collagen fibrils with tapered ends
following 4 hours' action of collagenase supplied by Dr. J. D.
MacLennan (Nandl; MacLeiiiian and Howes, ' 5 3 ) .
This paper describes the results of this same enzyme on
humaii skin and tendon and on calves' tendon. The qualitative
changes found under the electron microscope resemble the
stages of collagen synthesis reported in the literature. It is
possible, therefore, that enzyne break-down may shed light
on the visible component parts or building fragments of collagen. Concurrent quantitative chemical determinations will
be reported elsewhere. I n addition, the effects of various
* Arthritis and Rheuinatisni Foundation Research Fellow.
139
T E E A Y A TO X l IC A L RECORD, V U L . 119, S O 2
JUNE
1954
140
MADELINE li. IiEEC,H
preparations of trypsiii on collagen were studiecl under the
electron microscope.
MATERIALS AND MKTIIOL)
ColZagenuse. This was kindly supplied by Dr. J . D. AlacIminan and is obtained from CZ. 7iistoZyticzrm. It contains
some proteinase, but is one of the purest preparations available a t the moment, a s a crystalline form has yet to be made.
Auntan collageqz
Method 1 (unextracted). I n order to keep the condition
of tlie substrate as near as possible to that found in vivo the
usual chemical methods of extraction were avoided, since
such treatment may cause denaturation. Unfixed autopsy
dcrmis or Achilles tendon was homogenized in demineralized
water in a T a r i n g bleiidor in the cold room for 15 minutes.
The final temperature of the solution was 30°C. o r less in
each case. The suspension was then centrifuged for 20 minutes a t 2,000 RPhI at 4°C. and the sediment dried in a
vacuum. Twenty-five milligrarns of collagen were incubatecl
with collagenase (0.15 mg enzyme preparation nitrogen) and
0.05 ml penicillin and streptomycin mixture in a total volume
of 5 in1 phosphate buffer ( p H 7.3) for 18 hours a t 37°C. Drops
of this mixture were taken a t intervals during this incubation
period and placed on collodion-covered elcctron microscope
grids. They were allowed to dry, the excess buffer was removed by washing with demineralized water and they were
then shadowed with palladium. Preparations were examined
in the RCA EMU 50 KV elctron microscope. Controls were
treated in the same way including the antibiotic mixture hut
without enzyme.
Method 2 (extracted). Native collagen was extracted using
an abbreviated form of the second method described by Neuman ('49) employing 10% NaC1, N/15 NalHPO,, acetone,
alcohol and ether f o r one day each. Tlumps from tlie resulting
fibrous meshwork WCPC removed. Twenty-five niilligrams dry
COLLAGERASE A X D T R Y P S l S O N COLLAGES
141
iveiglit of the preparation were then incubated a s in method 1.
I n order to give a coniparable surface area for enzyme action
both extracted a i d unextracted substi~atcswere cut up into
small pieces before incubation.
Calves’ tendon
Samples of collagen from calves’ Achilles tendon were prepared according to method 1. A sample involving prolonged
extraction (Neuman, ’49) was kindly supplied by Dr. ,J. D.
Ogle.
RESULTS
CoiztroZs (table 1). After 18 hours’ incubation the tubes
were inspected macroscopically, and the clarity of the superiiate and the amount of disintegration of the substrate were
noted both before and after the tubes were shaken. The estractecl collagen controls were unchanged by shaking but
there were differences between unextracted adult and baby
skin. The original pieces of infant collagen floated a t tlie top
of a clear supernate, whereas the adult skin and calves’ tendon substrates were deposited a t the bottom of the tubes.
After moderate shaking the undigested unextracted adult
collagen became dispersed. Only very vigorous shaking produced sufficient, though minimal, disintegration of the infants’
substrate for suitable specimens to be obtained for electron
microscopy. The three-month-old child’s collagen was intermediate in its reaction between these two.
Obtaining satisfactory control specimens for electron
inicroscopic examination presented quite a problem : either
there was nothing on the grid or there were large lumps of
collagen that broke the collodion. I n spite of this, many
reasonably satisfactory preparations were obtained. I n contrast, the original material in water after blending and befoi-c
drying easily produced good specimens. Electron niicroscopy revealed a haphazard arrangeineiit of long, intertwining fibrils, the few visible ends being either square-cut or
obviously torn by blending, with asymmetrical projections
142
MADELINE I<. KEECH
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COLLAGENASE A N D TRYPSIN O X COLLAGEN
143
of component fibrils (fig. 1). Careful examination of a large
number of grids did not reveal any localized zones of narrowing in the fibrils or tactoids (vide infra). Fibers with
tapered ends were found fairly frequently in babies ’ skin and
occasionally in adult tissue but in no instance did they approach the number seen after 1-2 hours’ action by collagenase.
Collagenase (table 1)
-4Ictcroscopically there was a marked difference between
the degree of enzynie digestion of extracted and unextracted
substrates and between skin and tendon. Table 1 shows
that extracted calves ’ tendon was completely digested, while
the unextracted tendon was only about 85% digested a t the
end of 18 hours’ incubation. Adult human skin, obtained by
both methods of preparation, was only half digested, whereas
approximately 90% of the unextracted skin collagen from
three newborn infants had disappeared. The substrate f roni
a three-month-old child occupied an intermediate position
being about three-quarters digested. The supernates were
clear above extracted and slightly cloudy above the unextractecl substrates. After shaking the tubes containing the
extracted collagen there was little change in the appearance
of the agitated sediment or supernate, whereas the unextracted substrate became more disintegrated resulting in a
marked increase in the cloudiness of the supernate.
Electron microscopy of preparations of collagen incubated
with collagenase showed separation of the component fibrils,
many with tapered ends or localized narrowings in fiber
width, and tactoids (figs. 2 and 6). The tapered ends were
long and narrow and easily identified at fairly low magnification (3,000-5,000) and were quite unlike the “sheared
off” wide, triangular ends produced by the blendor. Localized narrowings in the width of the fibers varied in length
from some that occupied only two or three of the collagen
striations to others that were very long tenuous filaments
barely maintaining fiber continuity (figs. 3 and 4). There
was also a marked variation in the width or different com-
144
MADELINE K. KEECH
ponent fibrils (fig. 5). Tactoids (short lengths of collagen
tapered a t both ends) often occurred in clumps or groups
(fig. 7). Non-striated bi-tapered structures of much smaller
size were found only in extracted calves’ tendon after two
hours’ enzyme action (fig.8) although a careful search was
made in all the other substrates after one to 18 hours’ incubation. These structures are clearly different from the drying
patterns obtained from buffer. alone (fig. 9) or enzyme in
buffer (fig. 10).
A comparative study was made of the same samples of
skin and tendon prepared by both methods, treated with different concentrations of enzyme and incubated for varying
intervals. It was impossible to detect any differences in results between the extracted and unextracted substrate, differences in the enzyme concentration employed or in incubation periods of three as against 6 hours. All showed the same
degree of change under the electron microscope which consisted of tapering, localized fiber narrowings and tactoids.
However, concurrent chemical determinations of the total
nitrogen content of the filtrate following incubation showed
significant differences in comparing each of these three variables. Micro-Kjeldahl estimations revealed that more total
nitrogen was obtained from extracted than from unextracted
collagen per unit time, and that this total varied with increase in enzyme concentrations and period of incubation.
The reason that these chemical findings were not borne out
by electron microscopy is presumably the inability of the
latter to visualize the soluble products of hydrolysis. Further
details of the chemical analyses will be presented in a subsequent report.
Experimemts with t q p s i q t (table 1)
Co?%trols.Twenty-five milligrams of extracted and unestracted skin and tendon were incubated in 5 m l phosphate
buffer alone (pH7.3) for 18 hours a t 37°C. Macroscopically
there was no alteration in the substrate either before or
C O L L A G E N A S E A N D TRYPSIN O N C O L L A G E S
145
after shaking, and the supernate remained clear. S o changes
in the collagen fibrils were observed under the electron
microscope.
Trypsisz. M e t h o d . The same substrates were incubated
as above, but 1 m g of trypsin was added to each tube. A
highly purified preparation,2 4 different batches of L4rmour’s
crystallized trypsin and one of crude trypsin (DIFCO 1:250)
were all tested on different samples of the same collagen
preparations. S s crystallized trypsin contains up to 50%
MgS04, and this might have a possible effect on collagen,
both dialyzed trypsin and MgSO, alone (0.5 mg per tube)
were also studied.
Results. Illacroscopically. After 18 hours ’ incubation the
highly purified enzyme produced no change in the extracted
or unextracted collagen or supernates, either before or after
shaking. The other trypsin preparations gave no difference
from the controls before shaking, but after shaking there was
a significant difference between the tubes containing extracted
and unextracted substrates. The extracted collagen and supernate remained unaltered, whereas the unextracted collagen
was disintegrated and the supernate became cloudy (table 1).
Electron microscopy. All these enzyme preparations produced the same qualitative changes in collagen that were observed with collagenase, but minimal in degree (figs. 11 and
12). A few tapered ends and occasional localized fiber narrowings were found to the same extent with the highly purified and crystallized enzymes, but more frequently with
the crude preparation. Dialyzed trypsin produced the same
degree of change as undialyzed, and the MgSO, alone had 110
significant effect. Numerous helically coiled fibers as described by Gross ( ’51) were present in all batches of crystal“ Enzar ’’ (three-times crystallized and purified trypsin, lot. no. R550-13A)
kindly supplied by the Armour Laboratory Research Department. They state
t h a t it is dialyzed salt-free, and does not exhibit chymotryptic, ribonuclease or
desoxyribonuclease activity. The method of preparation should preclude the
presence of amylases, lipases and carboxypeptidase, hut it is possible that some
peptidases may be present.
146
MADELINE K. ICEECH
lized trypsin but were absent in the highly purified preparation.
Trypsirz plus trypsin khibitor. The same extracted and
unextracted substrates were incubated with 1mg crystallized
trypsin plus 1 4 m g soya bean trypsin inhibitor per tube.
After 18 hours’ incubation there was no macroscopic difference from the controls either before or after shaking. However, the same changes in the fibrils were observed in the
electron microscope as after the crystallized enzymes described above. The results were the same whether the trypsin
and inhibitor were allowed to remain in contact for 35 minutes
before addition of the collagen, or whether they were added
directly to the substrate. The potency of the soya bean was
verified by inhibition of a typical synthetic substrate for
trypsin, using the same batch of enzyme employed in these
experiments and under the same conditions.
Increclsimg amounts of highly purified trypsiri
(4‘
Enxar”)
Tubes containing the same batch of extracted skin collagen
as substrate were incubated as above with 12; and 25 times
the amount of “Enzar” previously used ( 5 mg and 10 mg
per 10 nig of collagen). Electron microscopy revealed a very
marked increase in the degree of fiber change noted in the
previous experiments using 1mg “Enzar” per 25 mg substrate. The fields were indistinguishable from those produced
by collagenase both morphologically and in degree (figs. 13
and 14).
Ezpcrirnerzts with proteiizase
Doctor J. D. MacLennan supplied some of the proteinase
contaminating the collagenase preparation. This proteinase
was chemically collagenase-free. Tubes containing the same
batch of substrate were incubated for 18 hours (with 5 mg
protease to 20 collagen) after which no macroscopic change
could be detected before or after shaking. Scanty tapered ends
and a few fiber narrowings and tactoids were seen under the
electron microscope, the degree of change being comparable
COLLAGENASF, A N D TBYPSIN O X COLLAGES
147
to that found with 1 m g of crystallized trypsin. If it is assumed that the collagenase contained as much as 50% proteinase, then the concentration of proteinase in the present
experiments would have represented an increase of 100,000
times over that used throughout this investigation. It is
concluded, therefore, that the proteinase itself has no action
011 collagen and that the changes noted were probably due
to a trace of contaminating collagenase in the proteinase.
DISCUSSION
Lacking precise knowledge as to the exact chemical action
of collagenase it is difficult to postulate the sequence of events,
but it would appear from these studies that the enzyme rapidly separates the component fibrils of the collagen bundles
and also attacks the individual fibrils both at their ends and
along their length, producing localized narrowings that elongate and finally break to form shorter components with tapered ends (figs 2-7). These symmetrically tapered ends are
striated to the tip and are quite unlike the square-cut or torn
ends produced by the action of the Waring blendor (fig. 1).
The action is identical on both extracted and unextracted skin
and tendon, the only difference being the very small tactoids
found after two hours’ enzyme action on extracted calves’
tendon (fig.8) which were not found with any other substrate
up to 18 hours’ incubation.
Vanamee and Porter (’51a) studied the solvation of collagen in rat tail tendon with acetic acid and its reconstitution
at rarying p H and salt concentrations. The sequence of this
fiber formation appeared to be well defined : lateral and longitudinal association of small proto-fibrils to form larger units
of the same shape, the process being repeated to form still
larger units and subsequently fibers. One per cent saline concentrations and pH ranging from 4.6 to 6.8 produced needleshaped crystals or tactoids showing the striated structure
characteristic of collagen. In fact, their figures 9 and 12
closely resemble our figure 8. Mercer (’52) discusses the possible methods of biosynthesis of fibers and postulates that the
148
MADELINE I<. KEECH
first step in the forniatioii of fibrous structures is the noiifibrous precursor of the proto-fibril which is transformed
into the proto-fibril (fibrillation). Three-dimensional arrays
of parallelized fibrils, i.e. the typical fibrous form, owe their
formation to lateral association of the proto-fibrils bp longrange colloidal forces. He cites keratin as an example of the
long thin cigar-shaped macrofibrils consisting of microfibrils
(proto-fibrils) and states that the shape of the macrofibrils
suggests that they are condensed tactoids produced by a
process such as described by Bernal ( ’40). It is coiiceivable
that the processes of synthesis and enzyme break-down may
resemble each other, and that the action of collagenase may
be one avenue of approach in finding the component parts 01’
building fragments of collagen. Should this assumption prove
correct, the findings reported in this paper would support
Randall’s suggestion (’52) that collagen fiber growth inay
take place by a process of pyramidal accretion in the manner
described by Mercer ( ’52) and Bernal ( ’40).
Trypsin has long been used to clean up collagen preparations for electron microscopic examination because it has
been assumed that it does not attack collagen. Tunbridge,
Tattersall, Hall, Astbury and Reed (’52) showed that ti*ypsin
does not affect normal collagen in human skin but removes the
misshapen collagen fibrils in abnormal skin in senile elastosis.
They state that pepsin “attacks collagen fibrils a t both sides
and ends, leaving tapering points and producing much amor0 enase
phous material. The action is similar to that of collag
on the fibrils of beef tendon.” However, they give no reference for the collagenase, and their two published electroii
micrographs demonstrating peptic action do not at all resemble the results reported in this paper. Apparently unpublished pictures of the earlier stages of peptic action
showed many short fibrils with tapered ends similar to those
illustrated here ( Astbury, personal communication). Vanamee and Porter (’51b) working on the solvation and reconstitution of collagen mention that partial break-down of the
COLLAGENASE A N D TRYPSIN O N COLLAGEK
149
fibers was accomplished by enzymatic digestion with trypsin,
but give no details.
As described above, 4 different batches of crystallized
trypsin, dialyzed trypsin and crude trypsin (DIFCO 1:250)
all produced cloudiness of the supernate and disintegration
of the unextracted substrate after shaking. I n contrast, the
tubes containing extracted collagen were unchanged. The
break-down effect was absent with the highly purified trypsin
and when crystallized trypsin and soya bean trypsin inhibitor
were incubated together. It was concluded that the unextracted substrate contained some non-collagenous protein
attacked by trypsin which was absent in the purified (extracted) preparation. The component producing this macroscopic change was apparently removed from the highly pnrified enzyme and was inhibited by soya bean. Northrop ( ’48)
states that soya bean trypsin inhibitor counteracts approximately an equal weight of pure trypsin, combining instantaneously to form an irreversible stoichoimetric compound.
The inhibitory effect is immediate and independent within
a wide rayge of solution pH.
I n the experiments described in this paper changes similar
to those found with collagenase were observed uncicr the
electron microscope with all the trypsin preparations used,
and were not inhibited by one and one-half times the weight
of soya bean trypsin inhibitor. Increasing the amount of
highly purified trypsin produced microscopic changes equal
in degree to that found with collagenase. These results suggest the presence of a cotaminant in the trypsin preparations, derived from the pancreas which is capable of attacking
collagen. The difficulty in obtaining an absolutely “pure”
substance by re-crystallization is well recognized. Kunitz
( ’ 3 5 ) found repeated crystallization necessary to obtain a
pure chynio-trypsinogen. McDonald ( ’48) not only had to
re-crystallize ribonuclease 5 times but also boil to rid it of
contaminating proteolytic enzymes. She points out that, although crystalline enzymes (or other protein) may be homo-
150
MADELINE K. KEECH
geneous electrophoretically and in the ultracentrifuge, they
may still contain minute quantities of impurities.
The macroscopic differences between the effects of both
collagenase and trypsin on extracted and unextracted substrate indicate a component in the latter (possibly fat, noncollagenous protein, hair follicles, etc.) which is not attacked
by collagenase. However, some of these impurities are attacked by crystallized trypsin to produce the greater disintegration and cloudy supernate as indicated in table 1.
SUMMARY
The action of collagenase on extracted and unextracted
collagen from human skin and Achilles tendon and on calves’
tendon demonstrated macroscopic differences between the
extracted and unextracted substrates. Abundant tapered
ends, localized fiber narrowings and tactoids were observed
under the electron microscope, and these changes were the
same irrespective of the source or method of preparation
of the substrate. However, very small component tactoids
were found only in extracted calves’ tendon.
Trypsin produced collagenase-like effects when viewed in
the electron microscope and these were not inhibited by soya
bean trypsin inhibitor. The changes were proportionate to
the amount of trypsin used, higher concentrations giving
a picture identical to that found with collagenase. The results suggest the presence of a contaminating pancreatic
enzyme capable of attacking collagen.
ACKNOWLEDGMENTS
I am deeply grateful to Doctors H. Bunting, W. G . Banfield, W. H. Gaylord, and J. D. Ogle without whose instruction, guidance, and help this work could not have been accomplished. I also wish to thank Dr. J. D. MacLennaii f o r
the collagenase, Dr. J. H. Milstone for the dialyzed trypsin,
trypsin inhibitor, and various batches of trypsin, and to the
Research Division of Armour and Company for the sample
of highly purified trypsin. My thanks are due to Dr. J. R.
Paul in whose department this work was done.
COLLAGENASE AND TRYPSIN O N COLLAGES
151
L I T E R A T U R E CITED
BANFIELD,
W. G. 1952 Occurrence of tapered collagen fibrils from human
sources with observations on mesenchymal neoplasms. Proc. Soe. Exp.
Biol. Med., 81: 658-660.
BEI(N.ZL,J. D. 1940 Structural units in cellular physiology. I n F. R. Moulton
(ed.), “Cell and Protoplasm,” Washington, D. C., Am. Assn. Ad>.
Sci., 199-205.
GROSS, J. 1951 Fiber formation in trgpsinogen solutioiis : a n electron optical
study. Proc. Soc. Exp. Biol. Med., 7 8 : 241-244.
KUNITZ,&I., AND J. H. NORTHRQP
1935 Crystalline chymo-trypsin and chymotrypsinagen. J. Gen. Physiol., 1s: 433-438.
MANDL,I., J. D. MACLENNAN
AND E. L. HOWES 1953 Isolation and characterization of proteinase and collagenase from C1. Histolyticum. J. Clin.
Invest., X X X I I : 1323.
MERCER,E. H. 1952 The biosyntliesis of fibers. Scientific Monthly, 75: 280-287.
MCDONALD,MARGARET
R. 1948 Proteolytic contaminants of crystalline ribonuclease. J. Gen. Physiol., 32: 33-37.
1948 A method of preparation of “ protease-f ree ’ ’ crystalline ribonuclease. Ibid., 3%: 39-42.
r\’EUAfAN, R. E. 1949 A comparative study of collagen and elastin. Univ. of
Chcinnati Thesis for Ph.D.
KODA,IT., AND R. W. G. WYCKOFF 3951 The electron microscopy of reprecipitated collagen. Biocliimica e t Biophysica Acta, 7 : 484-506.
NORTHROP,
J. H., M. KUNITZAND R. M. HEREIOTT 1948 “Crystalline Enzymes.”
Columbia Univ. Press, New York, 155.
RhNDALL, J. T.,R. D. R. m.4SER, S. JACKSON,
A. lr.W. hfART’IN AND A. C. T.
NORTH 1952 Aspects of collagen structure. Nature., I 6 9 : 1029-1033.
TUNBRIDGE,
R. E., R. N. TATTERSALL,
D. A. HALL, W. T. ASTBURYAND R. REED
1952 The fibrous structure of normal and abnormal human skin.
Clinical Sci., 8: 315-331.
TANAMEE,
P., AND I<. R. PORTER
1951a Observations with the electron microscope on the solvation and reconstitution of collagen. J. Exp. Med.,
94: 255-268.
1951b Observations on solvation and reconstitution of collagen.
Fed. Proc., 10: 263.
PLATE 1
EXPLANATION OF FIGURES
All collagen preparations shadowed w i t h palladium
1 Unextracted human Achilles tendon after 18 hours’ incubation in buffer only
showing blunt and torn ends of collagen fibrils due t o the Waring blendor.
X 27,500.
2 Unextracted adult hunian skin after three hours’ action by eollagenase. X
13,500.
3
Extracted adult human skin after 18 hours’ incubation with collagenase
showing two tenuous narrowings in one collagen fibril. X 20,000.
4
Unextracted adult human skin after three hours’ action by collagenase
illustrating three narrowings in a single collagen fibril. X 11,000.
5 Unextraeted calves’ tendon after one and a half hours’ mtion by eollagenase.
There is marked variation in the width of the different component fibrils,
some with tapered ends. X 10,500.
152
PI..\TE
153
1
G
Ciiestraeted adult l i u ~ i i a nskin : i f t r y 18 l i w r s ’ iiiciilmtion with col1;tgcn:isc.
r.
lliere are many short Ic~ngtlisof collagen tapwcil a t both ends (t:ictoid.;).
x 13,800.
154
COLLAGESASE A N D T R Y P S I S O X COLLAGEY
MA D E L I N E IC. P E P C H
155
PLATE 3
BXPI,AS.\TIOiV
O F FI GC R E S
i
Entraetcd adult liuinan skin a f t e r one :lid a half hours’ action 1): collayen:tsc
showing cluiiil)s of short lciigths of collagen tapered a t both ends (tsttoids) .
x 13.8010.
8
Extracted c;il.i-rs’ tendon :iftcr t n o hours’ action by collageixisc. The 1i:ic.kground contains iriany small, lion-striated, hi-tapered structui cs :iiid t h e e
proiiiiiieiit buffer crystals. The intact collagen fibril is ret:iined f o ~c o n parison in size. X 47,000.
9
Diluted phosplinte buffer.
10
X 14,500.
(’ollageansi~in l)liosplinte liuffer. X 6,000.
156
I’JATE 3
COI,I,AGENASE A S D TRTPSIN ON COLLAGES
M A D E L I N E h. hhL< H
157
PLATE 4
E S P L A S A T I O N O F FIGURFS
11 Uiicstracted adult liuinaii skin a f t e r 18 hours ' incubntioii iritli 1lug purified
trypsiii (Armour). There :ire tapcred ends and fiber ii:irronings siiiiikir to
those fouiid with collagcnase. X 18,300.
12
Extracted adult human skin after 18 houri' incubation '11 itli 1iiig cr>-stnllizctl
tr) p i n . The end of tlie collagen fibril is surrounded 11.r the tlrrwds :ind
filaments present in solutioiis of crystallized trypsin iis dcmonstr:ited by
('51). X 18,650.
13 E i t r a c t e d human adult skill after 18 hours' incubation with purified t r y p i n
(Ariiiour) a t n couceiitrntioii of 12.5 iiig per '75 iiig eollngeii. Tlicire :ire
7 iiarrowiiigs in a single collagen fibril. x 16,500.
14 Same as figure 13. The groups of tactoids are similar to those i1lustr:ited
in figure 7 , X 15,800.
158
PLATE
COLLAGESASE AXD TRPPSIN ON C O I J I A G E S
MADl(:l,lNE
I(.
JiBECH
159
4
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