265.
'STA IUS O f CONNECT I VE T I'SSUE RESEARCH
J.
WALTER 01 FFEE
FOUNDATION
...........................................
AMERICAN MEAT
INSTITUTE
Introduction
Since t h i s conference is primarily concerned w i t h the q u a l i t y of
meat I w i l l attempt t o emphasize those connective t i s s u e f a c t o r s which
exist i n muscle and which may contribute t o t h e i r physical properties. For
t h i s reason consideration of the various c e l l u l a r components w i l l be omitted.
The p e r f o m c e of these c e l l s under t h e influence of i r r i t a n t s , n u t r i t i o n a l
f a c t o r s and hormones is a topic unto i t s e l f . In view o f time l i m i t a t i o n s I
will confine my discussion t o t h r e e p r i n c i p a l areas (1)mucopolysaccharides
(2) e l a s t i n , and (3) collagen.
Mucopolysaccharides
Mucopolysaccharides are those macromolecules which contain amino
sugars. In recent years rapid progress has been m,de indicating these substances t o be highly complex, and d i f f e r i n g i n composition from organ t o
organ. I n f a c t , most t i s s u e s contain a mixture of t w o o r three d i f f e r e n t
polysaccharides.
Enumeration of the detailed chemistry of these substances does not
s u i t our present purpose but a number of icrportant features useful i n d i f f e r e n t i a t i n g between species have been tabulated (Table I). In addition t o
t h e features summarized i n t h i s table, it is a l s o i n t e r e s t i n g t o note that
most o f the glycosidic linkages a r e i n beta configuration and that they
appear t o be 1 3 o r 1 4. Although d e t a i l s of gross s t r u c t u r e a r e lacking, these molecules appear t o be chain-like with l i t t l e o r no branching.
These chains tend t o c o i l i n t o globular form.
-
-
-
mst of the research on mucopolysaccharides has been performed on
connective tissue r i c h sources. While these carbohydrates are frequently
seen providing an imbedding matrix f o r proteinaceous fibers they a r e very
often i n chemical combination with a v a r i e t y of proteins and peptides. For
example, c a r t i l a g e i s r i c h i n chondroitin s u l f a t e A, a f r a c t i o n of which i s
found i n firm combination with a non-collagenous protein. Chondroitin
s u l f a t e s A and C a r e found i n developing connective t i s s u e . Their tenure
is t r a n s i e n t , however, and mature t i s s u e of loose texture i s more l i k e l y t o
be rich i n hyaluronic acid which serves as a lubricant f o r strands of
collagen and e l a s t i n .
The task of measuring these substances i n muscle both quantitat i v e l y and q u a l i t a t i v e l y is a d i f f i c u l t one; establishing t h e i r relationship with t h e proteins i s s t i l l another. Yet we need t h i s information as
w e delve f o r a U e r understanding of muscle t i s s u e .
Elastin
Progress i n the understanding of the chemistry of e l a s t i n has
developed slowly. Very pure preparations have been prepared and the amino
266.
acid composition detemined. More than half of the amino acid residues a m
glycine and alanine while l e s s than t e n percent of t h e residues have polar
s i d e chains. This deficiency of charged groups.accounts f o r the lack of
swelling i n a l k a l i o r acid. Elastin i s exceedingly insoluble i n neutral
solvents and has been shown .to withstand heating t o 1 2 0 O C . f o r six hours
with l i t t l e o r no degradation. However, solutions of boric acid a t 1 0 0 ° C ,
bring about p a r t i a l hydrolysis i n t o two principal components, .& - e l a s t i n
( m l e c u l a r weight (60 80,000) and 8 - e l a s t i n (molecular weight 6000)). The
higher molecular weight f r a c t i o n is capable of forming a g e l which becomes
increasingly s t a b l e with age.
-
Analysis by X-ray d i f f r a c t i o n and electron microscopy have f a i l e d
t o demonstrate well defined patterns of periodicity. It has been shown by
Ramachandran that collagen p a r t i a l l y denatured by b r i e f exposure t o lyotropic
agents (e.g. CaC12) y i e l d s an X-ray p a t t e r n similar t o t h a t of e l a s t i n . This
l e d Partridge t o suggest t h a t a three stranded s t r u c t u r e s i m i l a r t o that of
collagen may be indicated by such behavior. The most plausible hypothesis
proposed thus f a r is a l s o from Partridge and Davis. They assume t h a t e l a s t i n
is cliemically homogeneous and consists of randomly coiled chains approximately oriented wfth the f i b e r axis. Future development of o u r knowledge
regarding actual s t r u c t u r e w i l l pmbably come from refinements i n techniques
i n v o l v i n g 6 and B-elastin.
-
Collagen
.
.
Many of t h e outstanding adtances i n protein cheinistry have been
used i n a r r i v i n g a t our present s t a t e of knowledge regarding collagen.
When i n 1948 Orekhovich e t a l . reported the solubilization of collagen fratt i o n s i n acid buffers and proposed that the soluble f r a c t i o n s represented
procollagen, a large number of new workers entered t h e f i e l d . Fraction6 of
soluble collagens wen? found i n weakly alkaline solution, neutral salt
solutions, and i n solutions of l y o t m p i c agents. As knowledge progressed
it became apparent t h a t t h e true collagen fiber precursor, i . e . t h e single
collagen molecule composed only a small f r a c t i o n of the t o t a l collagen
present i n a c i d e x t r a c t s . In f a c t varying states of aggregation appear i n
all the collagen solutions and prolonged ultracentrifugation i s required t o
remove dimers, trimers, and o t h e r polymeric forms. It became necessary t o
use t h e term tropocollagen t o describe the mnomeric form. Examination of
solutions of tropocollagen by physical chemical methods indicated t h a t t h e
molecule behaves as a r i g i d rod about 14 A wide and 2800 A long. Techniques
of electron microscopy developed by Hall p e m i t t e d the confirmation of these
dimensions.
--
These findings with rega-rd t o monomeric collagen aided g r e a t l y i n
t h e i n t e r p r e t a t i o n of electron photomicrographs. For a nuiber of years
beautiful micrographs have been obtainable f r o m stained native fibers.
By s u i t a b l e manipulation of collagen ~ o l u t i o n sa v a r i e t y of
(Figure
forma can be precipitated including a fibrous form i d e n t i c a l w i t h the native
f i b e r seen i n t i s s u e , One of t h e most i n t e r e s t i n g p r e c i p i t a t e s , obtained
by adding ATP t o acid solutions of collagen, was termed segment long spacing
o r SLS. (Figure 2 ) . As the r e s u l t of some r e a l l y b r i l l i a n t research among
several gmups of workers, it has been determined t h & t t h i s SLS form represents l a t e r a l aggregates of monomeric collagen with t h e i r poles i n r e g i s t e r .
1 ) b
267.
Selective staining w i t h acid binding and base binding groups has revealed
t h a t t h e a c i d i c and basic groups occur i n the same regions of the molecule
and are responsible f o r the cllaracteristic bands appearing i n the photomicrographs (Figure 3). These bands of charged groups are responsible f o r
the l a t e r a l orientation of the molecules within t h e f i b e r .
It remained t o r e l a t e the SLS pattern t o that of native fibers.
While the molecule was shown t o be 2800 A long t h e f i b e r exhibited a major
p e r i o d i c i t y of about 700 A, This suggested t h a t the molecules i n the f i b e r
were i n a quarter staggered array. This was proved by photographic means.
An SLS photomicrograph was employed i n a multiple exposure technque i n
which each exposure was displaced one-quarter of the p a t t e r n length. (Figu r e 4)
The composite revealed a t y p i c a l native f i b e r pattern! Furthermore, it is evident from these patterns as well as o t h e r d a t a t h a t l i n e a r
polymerization is a head t o t a i l type.
For several years now it has been recognized that the X-ray data
a r e most probably explained by a three stranded helix s t r u c t u r e . When a
solution of collagen i s wanned t o i t s denaturation temperature the strands
unwind and tend t o d i s s o c i a t e i n t o w h a t is termed parent g e l a t i n . Ecaminat i o n of these solutions by physical methods has revealed t h a t as many as
three species may be present, and they correspond t o one, two, and three
stranded structures. The three stranded s t r u c t u r e can, by s u i t a b l e raanipul a t i o n , be renatured t o form fibrous collagen. h r t h e r evidence has indicated t h a t the bonds between t h e multistranded gelatins are covalent i n
character, t h a t e s t e r bonds involving glutamic acid a r e important and that
carbohydrates a r e a l s o involved.
One of the important observations made by workers engaged i n the
extraction of soluble collagen related y i e l d of t h i s material t o t h e age of
the animal. Connective t i s s u e s of very young, rapidly growing animals gave
a y i e l d several f o l d that obtainable from mature t i s s u e . Several i n v e s t i gators, p a r t i c u l a r l y Veis, have f e l t t h a t the increasing s t a b i l i t y of
mature collagen is due t o the development of intermolecular covalent bonds.
This point of view appeared plausible t o u s when we compared the action of
CaCl2 solutions a t 2 O C. on calfskin collagen with t h a t from steerhide
(Figure 4 ) . The reagent concentrations used would overcome hydrogen bonding e f f e c t s a s well as e l e c t r o s t a t i c a t t r a c t i o n s . Increasing t h e extraction
temperature t o about 30' C. caused lower concentrations of CaCl2 t o have the
same extracting efficiency a6 the higher concentrations but d i d not increase
the amunt of collagen extracted. The wide range between the young and the
mature t i s s u e s suggests an increasing gradient i n numbers of intermolecular
bonds with age. Connective t i s s u e s t a b i l i t y (which may be influenced by
genetic f a c t o r s a s well as age) can be an important f a c t o r i n meat q u a l i t y .
Another area which has a t t r a c t e d our a t t e n t i o n has been the action
of proteolytic enzymes on collagen. With the exception of b a c t e r i a l
collagenase, native collagen resists the action of a l l known proteases a t
neutral pH. Upon denaturation by heat o r chemical agents, however, t h e
collagen becomes generally susceptible t o a t t a c k by a v a r i e t y of proteol y t i c enzymes and is rapidly dispersed. The denaturation temperature of
bovine collagen i n water is about 141O F. but i n physiological s a l i n e it
drops t o 133O F. Below pH 6.5 t h e denaturation temperature gradually dec l i n e s . Sliwinski showed t h a t the proteolytic enzymes of muscle survive
120' F. and are inactivated a t 140' F. -i n situ. This assembly of f a c t s
268.
provokes a number of i n t e r e s t i n g questions. Do t h e p r o t e o l y t i c enzymes of
muscle a t t a c k collagen during cooking? To what extent a r e these enzymes
f r e e d t o come i n contact with collagen post-mortem? Does t h e tenderness of
meat have anything t o do with the amount of p r o t e o l y t i c e n z p e i n t h a t
p a r t i c u l a r cut ?
Table I
Characteristics of Mucopolysaccharides.
Species
Components
IIydrolys is by
Physical properties
Hyaluronidase
A*
Hyaluronic
acid
glucuronic acid
Chondroitin
sulfate A
glucuronic acid
Chond roit in
iduronic acid
sulfate B
Chondro it i n
sulfate C
I
_
Chondroitin
+
N- Acglucosamine
4.
+
-
-
-
+
-
(N- Ac galactosamine
( s u l f a t e on C -6
(M- Ac galactosamine
( s u l f a t e on C-6
glucuronic acid
-
N- Ac galactosamine
glucuronic acid
galactose
(4
-
M, W.
8x106
D
30'
(4
-
50'
(4
-
20°
M. W.
<A
(4
-
+
D
M. W. 15-26Gx103
C a s a l t insoluble
i n 20;; EXOH
D
D
Ca s a l t soluble
i n 40-50$ &OH
+
.
-
(4
-
-
(H
D
D
(N- Ac glucosamine
(sulfate
Heparat i n
sulfate
glucuronic acid
(N- Ac glucosamine
(N- s u l f a t e
(0- s u l f a t e
*
A. T e s t i c u l a r
B. Microbial
700
-
(4
4-
N- Ac galactosamine
Qratosulfate
B*
1
D
+
210
269.
REFERENCES
Page, I. H. (Ed.),Connective Tissue, Thrombosis, and Atheroscleroeis (Academic
Press, New York and London), 1959.
Wolstenholme, G , E. W., and O'Connor, M. (Ws.),Ciba Foundation Symposium
on t h e Chemistry and Biology of Collagen ( L i t t l e , Brown and Company,
Boston) 1958.
Asboe-Hansen, G. (Ed.), Connective Tissue i n Health and Disease (Philosophic a l Library, New York) 1957.
OtFlaherty, F., Roddy W, T., and Lollar, R. M. (ms.),Chemistry and Technology of Leather, Vol. 1 (Reinhold Publishing Corporation, New York)
1956.
Stainsby, G. (EU..), Recent Advances i n G e l a t i n and Glue Research (Fkrgarnon
PTess, New York, London, Paris, IDSAngelee) 1958.
Harrington, W,, and Von Hippel, P. HI, "The Structure of Collagen and
Gelatin," Advances i n Protein Chemistry
Figure 1,
Electron micrograph of n a t i v e collagen f i b e r s t a i n e d with phosphotungstic
acid.
Figure 2.
Segment long space (SLS) collagen s t a i n e d with phosphotungstic acid.
Figure 3 .
SLS collagen s t a i n e d with phosphotungstic acid matched with SLS collagen
s t a i n e d with uranyl a c e t a t e . Coincident bands reveal juxtaposition of a c i d
and basic amino acid side chains i n t h e oriented molecule.
Figure 4.
Photographic synthesis of native fiber p a t t e r n by repeated overlapping exposures of SLS p a t t e r n .
-----------(Applause )