Download Characterization and Determination of Muscle Connective Tissue

242.
C H A R A C T E R I Z A T I O N A N 0 OETERMINA T l O N OF MUSCLE
CONtdEC T I
YE T I'SSUE COMPONNZN TS
The muscle connective t i s s u e components have long been linked
with meat tenderness and f o r t h i s reason t h e i r determination and, m o r e re-
cently, t h e i r characterization have received considerable attention from
meat researchers. Already i n the 1930rs, Helser, Bull, Mitchell, Hamilton,
and others reported a negative correlation between the quantity of connect i v e tissue and meat tenderness. However, these early studies were handicapped by t h e l a c k of adequate techniques f o r t h e quantitative determination
of connective tissue. In 1941, I ~ w r yand comrkera (15) published a technique f o r collagen and e l a s t i n determination Based on the i n s o l u b i l i t y or"
these proteins. Neuman and Logan (22) presented the f i r s t adequate method
f o r hydroxyproline determination i n 1950. Although both of these techniques
are s t i l l widely used today i n modified forms, several studies have shown
that neither i s completely s a t i s f a c t o r y f o r t h e determination of connective
t i s s u e i n meat (1, 1 2 , 16, 39).
bbre recently, the chemical makeup and t h e three-dimensional
structure of connective tissue components have been extensively investigated (6). These studies have revealed mrked differences i n the chemical
s t r u c t u r e of connective t i s s u e isolated f r o m animals of d i f f e r e n t ages. It
i o not unreasonable t o wsume that the three-dimensional structure as w e l l
as the amounts of the connective tissue c o r n n e n t s might be involved i n
meat tenderness. In fact, t h i s disregard of the three-dimensional struct u r e may be responsible f o r some of the conflicting reports regarding t h e
importance of connective tissue i n meat tenderness.
I n order t o make an i n t e l l i g e n t analysis of the problems of connective t i s s u e determination and characterization, it is necessary t o be
familiar with its chemical composition and the chemical and physical properties of its components. Connective t i s s u e consists of three morphologic a l l y d i s t i n c t constituents.
h i s paper was written during t h e tenure of a Predoctoral Fellowship from
the Division of General Medical Sciences, United States R b l i c Health
Service.
243.
Slide 1
Constituents of Connective Tissue
Cells
1. Fibroblasts
(embryonic c e l l s ) ,
---Ground Substance
---*
1. Hyaluronic a c i d
2.
Fat c e l l s
2.
Keratosulfate
3.
blast c e l l s
3. Chondroitin s u l f a t e A
4.
Ivlacrophages
4.
Chondroitin s u l f a t e B
5.
Mesenchymal
5.
Chondroitin s u l f a t e C
cells
6.
Heparin
(undifferentiated)
7,
Reparitin’ SUI-fate
Proteins
1, Collagen
2.
Elastin
3,
I?e-ticulin
--
The connective t i s s u e c e l l s w i l l not be discussed since D r .
Nullins has already discussed these components, p a r t i c u l a r l y t h e mast c e l l a ,
a t l a s t year’s Reciprocal Meats Conference ( 2 0 ) .
Although up t o now t h e gmund aubstance has not been extensively
investigated, recent s t u d i e s have provided ~ o m eevidence on t h e chemical
s t r u c t u r e of t h e mucopolysaccharides found i n t h e ground substance (19).
Slide 2
bbcopolysaccharides of Connective Tissue
Mucopolysaccharide
Hyaluronic a c i d
Constituents
N-acetylglucosamine (1)
Glucuronic a c i d (1)
Keratosulf a t e
N-acetylglucosamine (1)
Galactose (1)
Sulfate (1)
Hepa r i n
Glucosamine (1)
Glucuronic a c i d (1)
Sulfate (3)
Heparitin s u l f a t e
Glucosamine (1)
Glucuronic a c i d (1)
Sulfate (1)
Linkage
(1-+3) glucuronidoglucosamine
(1-+4) glucosarninidoglucuronic a c i d
Slide 3
Mucopolysaccharides from Connective Tissue
Mucopolysaccharide
Chondroitin sulfate A
Constituents
N-acetylgalactosamine
Glucuronic acid (1)
Chondroitin sulfate B
Sulfate (1)
-
Linkage
(1-+3) glucuronidogalactoeamine
(1-4)
galactosaminidoglucuronic acid
N-acetylgalactosamine
( 1 4 3 ) iduronidogalactosarnine
L-idumnic acid (1)
(1- 4) galactosaminido
iduronic acid
-
Sulfate (1)
Chondroitin sulfate C
N-acetylgalactoaamine
Glucuronic acid (1)
(l-+ 3) glucuronidogalactosamine
( 1 44) wactosaminidoglucuronic acid
Sulfate (1)
In vivo, these mcopolysaccharides are presumed to be complexed
with a noncollagenous protein to form various mucoprotein substances (25).
Although these mucoproteins will not be discussed firther because of the
dearth of information concerning their role in meat tenderness, their importance should not be underestimated. Further studies on the relationship
between these components and meat tenderness are indicated by McIntosh's
recent report of a negative correlation between the mucoprotein content and
tenderness of skeletal muscle (21). The ratios of the contents of the different mucopolysaccharides components have also been shown to differ among
different sources and this m y have aome importance in meat tenderness.
While the three fibrous connective tissue proteins belong to the
insoluble class of proteins called the scleroproteins, their chemical behavior is much more complex than this classification might indicate.
245,
Slide 4
Proteins from Connective Tissue
Collagen
1. Banded under t h e
EM-700
8
Raqtin
1, Not banded under the
electron microscope
. .
2.
Not s t r e t c h e d 1% by
1,000 times i t s
own weight
3.
Solubilized t o
g e l a t i n by hot
aqueous e x t r a c t i o n
3.
4.
Not hydrolyzed by
trypsin
4, Not hydrolyzed by
trypsin
5.
Eydrolyzed by
collagenase,
f i c i n , papain, and
some b a c t e r i a l
enzymes
5.
6.
Crystalline
structure
6. Amorphous s t r u c t u r e
2.
Reticulin
1. Precollagen fibers
Easily s t r e t c h e d
2.
Highly branched
N o t a f f e c t e d by hot
aqueous extraction
3.
P a r t i a l l y hydrolyzed by t r y p s i n
4.
Contains some
.
lipid
Hydrolyzed by
elastase, f i c i n ,
and papain
Reticulin i s a poorly characterized p r o t e i n which g r e a t l y resembles collagen, although it i s not as widespread. Also, it possesses d i f f e r e n t s t a i n i n g properties than collagen, contains a tightly-bound, myristic
a c i d - r i c h Z.ipid, a d some kinds of it appear t o be r e s i s t a n t t o collagenase.
Since it i s not present i n l a r g e amounts, it should not be a major f a c t o r i n
meat tenderness.
Ivbscle i s reported t o contain about 1/3 as much e l a s t i n as
collagen. E l a s t i n i s n ' t markedly affected by hot aqueous solvents and thus,
it shou3.d play an important r o l e i n meat tenderness. The amino a c i d composition of e l a s t i n may explain i t s i n s o l u b i l i t y i n aqueous solvents since
it contains over 90s nonpolar amino acids by weight. It is similar t o
collagen i n i t s glycine and proline content, containing 27% and 13$ of these
two amino acids respectively. These nonpolar amino acids together w i t h the
presence of a l i p i d which appears t o be t i g h t l y bound t o t h e e l a s t i n molecule make it q u i t e r e f r a c t o r y t o aqueous solvents. This same unique amino
a c i d composition may a l s o explain t h e r e s i s t a n c e of e l a s t i n t o many enzymes.
Very l i t t l e is known about the three-dimensional s t r u c t u r e of elastin due
t o t h e d i f f i c u l t y of obtaining good x-ray d i f f r a c t i o n p a t t e r n s . Recent
investigation using elastase as a probe i n t o t h e chemical s t r u c t u r e of
e l a s t i n may help t o c l a r i f y t h e r o l e of t h i s p r o t e i n i n meat tenderness.
Collagen, l i k e e l a s t i n , has an unique amino a c i d composition. It
is the only p r o t e i n known t o contain hydroxylysine, containing 6-7 residues
26-6.
of t h i s amino acid per 1,000 amino acid residues. Also, it is t h e o n l y
protein which contains an appreciable amount of hydroxyproline although
e l a s t i n contains 2$ by weight of t h i s amino acid. Slide 5 shows t h e seven
most abundant amino acids i n collagen.
Slide 5
Amino Acid Composition of Collagen
Glycine 33.5$, proline 13 .l$, alanine 10.5$, hydroxyproline 9.5$,
glutamic acid 7.1$, aspartic acid 4.75 and arginine 4.55
Although over 70$ of the amino acids i n collagen a r e nonpolar i n
character, the high dibasic acid and arginine content prevent collagen from
being a s nonpolar as e l a s t i n , Very few aromatic o r s u l f u r containing amino
acids a r e found i n collagen and tryptophan is absent e n t i r e l y . This l a t t e r
f a c t has been used as a test f o r the purity of collagen preparations.
Although collagen has t r a d i t i o n a l l y been thought t o be an insoluble
protein, small fractions of it can be extracted by aqueous buffers. These
fractions have been termed neutral salt 60lUble or acid soluble collagen
depending upon whether pH 7 phosphate buffer o r pH 4 c i t r a t e o r acetate
buffers were used i n t h e i r extraction, physicochemical. studies show these
fractions t o be monodisperse containing protein molecules with a molecular
weight of nearly 350,000. These protein molecules a r e thought t o re resent
monomers and have been named tropocollagen. They are 2800 long
i n diameter appearing i n the form of a long rod i n the native
fl
state.
I
Electron micrograph of tropocollagen molecules magnified 100,000 X
R e c t r o n micrograph of heat denatured tropocollagen
m l e c u l e s magnified 100,000 X
I
Slide 8
Left-handed h e l i c a l peptide chains
I
A
I
Slide 9
Formation of the collagen I and I1 structures by the coiling of the t h r e e
left-handed h e l i c a l peptide chains into a right-handed super c o i l
1
247.
1
I
S l i d e 10
The t r i p l e h e l i x
I
Slide U.
Cross-sectional view of the t r i p l e h e l i x
I
When t h e peptide chains are coiled i n t o a t r i p l e h e l i x of t h i s
type, every t h i r d position along an individual peptide chain is i d e n t i c a l ;
t h a t is, it has an i d e n t i c a l environment. These positions may be numbered
sequentially 1, 2, and 3 . Because of conditions of s t e r i c hindrance, there
are c e r t a i n r e s t r i c t i o n s concerning t h e nature of the amino acids which may
occupy these positions.
Slide 1 2
The Possible Positions of Side Chains
Position
I
2
H bonding of
t h e OH group
of Hy-pro i n
position 3
Collagen I
Undef ormed
Def o rtned
Gly o n l y
Other residues
possible except
Pro and Eypro
Collagen I1
Gly only
Any residue
A n y residue
Any residue
G l y only
Any residue
Any residue
Bonds t o a
neighboring
chain within
t h e group of 3
Cannot bond
within t h e
group of 3
a t i c k s out
radially
-"
except
Val and I l e u
-
The t r i p e p t i d e gly-pro-hypro has been found t o be t h e predominant
product of a collagenase d i g e s t , so s t r u c t u r e I1 i s the one favored by most
investigators since it can a c c o m d a t e t h i s sequence of amino acids with t h e
l e a s t s t r a i n . Sequences of amino a c i d s a s long as 24 residues which cont a i n no hydroxyproline and o n l y s m a l l amounts of proline have a l s o been
found i n these collagenase d i g e s t s . Therefore, it has been proposed t h a t
t h e amino a c i d composition of t h e tropocollagen niolecule is not uniform
along i t s length, but instead t h e r e e x i s t areas of higli proline, glycine,
alanine, and hydroxyproline content where every t h i r d amino acid i s glycine
and t h e sequence gly-pro-hypro predominates. These areas possess t h e
" c r y s t a l l i n e " t r i p l e helix s t r u c t u r e and a r e attacked by collegenase. The
narrow s p e c i f i c i t y of collagenase requires t h a t every t h i r d amino a c i d be
glycine, although alanine may be s u b s t i t u t e d f o r glycine with some decrease
i n t h e rate of hydrolysis.
248.
Slide 13
I
Specificity of Collagenase
I(
-
Pm
R1
Pro
- R~ + R~ - ~ r o
:Proline
=
% =
o r hydroxyproline
Any amino acid
Glycine o r alanine
Between these ordered c r y s t a l l i n e areas, there a r e areas high i n
glutamic and a s p a r t i c acid. These areas don't possess the t r i p l e h e l i x
s t r u c t u r e but a r e i n t h e form of a more extended chain having a g r e a t e r
amount of disorder. Another s t r u c t u r a l f e a t u r e of the tropocollagen molecule i s t h e presence of a " t a i l " a t e i t h e r end a s t h e r e s u l t of the project i o n of one of t h e three peptide chains past t h e other two a t t h e end of t h e
molecule. This t a i l is high i n arginine, is hydrolyzed by trypsin, and is
responsible f o r the aggregation of tropocpllagen t o f o m collagen f i b e r s ,
Investigators were p u z z l a f o r some tinie by t h e mechanism whereby
these tropocollagen molecules 2800 A long aggregated t o form collagen f i b e r s
with a 700 A periodic cross-banded structure. The discovery of two new
types of collagen, t h e "fibrous long spacing" and the "segment long spacing"
l e d t o the solution o f t h i s problem.
1
Slide 14
Normal collagen under t h e electron m i c r o s c o p e l
Fibrous long spacing collagen under the electron microscope
.
1
Slide 1 6
~~
Segment long-spacing collagen under the electron microscope
I
I
Slide 1 7
I
Aggregation of Tropocollagen
Normal collagen is formed by tropocollagen molecules aggregating
head t o t a i l successive chains overlapping by 1/4 of t h e i r length t o give a
700 8 period. Fibrous long-spacing collagen is formed by tropocollagen
molecules aggregating head t o t a i l with a l t e r n a t e ch i n s arranged i n an
a n t i p a r a l l e l fashion with no overlap t o give a 2800 period. Segment longspacing collagen i s formed by the tropocollagen molecules aggregating side
t o side but not l i n e a r l y .
It
249.
The interaction of t h e aggregated tropocollagen molecules w i t h
e l a s t i n and t h e mucoprotein ground substance forms the material we know as
connective t i s s u e .
The methods used t o study these connective t i s s u e components may
be broadly c l a s s i f i e d i n t o those used f o r the study of t h e three-dimensional
s t r u c t u r e and those used f o r the quantitative determination of these components. Many d i f f e r e n t physicochemical techniques are available for t h e
study of three-dimensional s t r u c t u r e but most of them require the i s o l a t i o n
of the substance under study i n a pure and unaltered state. Obtaining pure
and undegraded connective t i s s u e components from muscle t i s s u e i s obviously
a very d i f f i c u l t t a s k and f o r t h i s reason, many of these techniques have
not yet been used t o s t u d y t h e muscle connective t i s s u e components.
Slide 18
Methods f o r Three-dimensional Structure
1. X-ray d i f f r a c t i o n
2.
Melting curves
3.
Gelation
4.
Electron microscope
5.
Spectroscopy
6.
Optical rotation
7.
Dielectric dispersion
8.
Collagenase and other enzymes
This s l i d e presents only a few of the many d i f f e r e n t methods
available f o r t h e study of three-dimensional s t r u c t u r e .
The use of x-ray d i f f r a c t i o n i n the study of the t r i p l e h e l i c a l
s t r u c t u r e of collagen has already been mentioned. Since Dr. Krimm has discussed t h i s technique i n some d e t a i l , nothing more w i l l be s a i d about it now.
By melting curves, I am r e f e r r i n g t o e i t h e r one o f t w o kinds of'
s t u d i e s . The temperature of a solution of tmpocollagen molecules may be
gradually raised and changes i n t h e v i s c o s i t y and o p t i c a l rotation of the
solution followed as a function of temperature, o r collagen f i b e r s may be
substituted i n place of the tropocollagen molecules and t h e temperature of
the l i q u i d i n which t h e f i b e r s a r e immersed then slowly raised u n t i l the
f i b e r contracts t o 1/3 of i t s o r i g i n a l length. T h i s temperature of cont r a c t u r e i s known a s the shrinkage temperature, T,, and is quite characteri s t i c of a p a r t i c u l a r collagen sample. Both of these approaches may give
some indication of t h e degree and strength of cross l i n k i n g i n t h e collagen
molecules o r fibers
.
250.
Gelation studies have been discussed i n a number of recent s t u d i e s
He has extracted g e l a t i n from collagen
samples by heating a t 60' C. f o r 1 t o 2 hours i n pH G buffers. The g e l a t i n s
obtained can be separated i n t o several f a i r l y homogeneous f r a c t i o n s of d i f f e r i n g molecular weight. The r e l a t i v e proportions of these f r a c t i o n s may
again provide some i n f o m a t i o n concerning cross linking.
by Veis (32, 33, 34, 35, 36, 37).
Bob Caesens has already discussed some aspects of the use of t h e
electron microscope i n the study of protein s t r u c t u r e . Its use t o obtain a
v i s u a l representation of t h e molecule is an obvious one.
S2ectroscopy includes t h e use of infrared, u l t r a v i o l e t and nuclear
magnetic resonance spectra as well a s various modifications of these techniques. n e c t r o n spin resonance has a l s o been found t o be a useful t o o l f o r
studying the formation of free r a d i c a l s during enzyme reactions. These
techniques, p a r t i c u l a r l y t h e i n f r a r e d and nuclear magnetic resonance spectra,
provide a valuable t o o l f o r t h e study of t h e types o f bonds and chemical
groups which are present i n a molecule as w e l l as the o r i e n t a t i o n of these
groups.
Optical r o t a t i o n is a useful method f o r studying changes i n t h e
h e l i c a l content of a molecule. It is p a r t i c u l a r l y valuable f o r followfng
changes i n molecular s t r u c t u r e due t o the application of various treatments.
D i e l e c t r i c dispersion i s a t o o l which has not been extensively used
i n t h e study of protein stru-cture. It i s defined as the v a r i a t i o n of the
d i e l e c t r i c constant with t h e frequency of t h e e l e c t r i c f i e l d and may give
some information about t h e r o t a t i o n a l freedom i n t h e proteih molecule.
The use of collagenase and o t h e r enzymes may provide u s with i n formation regarding t h e amino a c i d sequence of a protein as w e l l as informat i o n on i t s three-d.imensiona1 s t r u c t u r e . The k i n e t i c s of enzymic digestion
may be markedly altered by cross linking, h e l i c a l coiling, or any o t h e r
phenomena which may render susceptible bonds i n t h e protein less accessible
t o t h e enzyme (2, 9, 23, 31, 3 8 ) .
A t l i s c o n s i n , we have recently kmbarked upon a study using melting
curves, gelation, and collagenase digestion t o study connective t i s s u e samples f r o m anfmals of d i f f e r e n t ages. These techniques have revealed s i g n i f i cant differences among connective t i s s u e samples i s o l a t e d from t h e d i f f e r e n t
age g r o u p .
The methods used f o r t h e q u a n t i t a t i v e determination of collagen
and e l a s t i n may be c l a s s i f i e d i n t o two d i f f e r e n t groups depending on
whetner they are based upon s o l u b i l i t y differences o r upon hydroxypnline
determination. (I am here confining the discussion t o t h e chemical determination of collagen and e l a s t i n since t h e subsequent paper will present t h e
various aspects of the h i s t o l o g i c a l determination of these components i n
d e t a i l ) . Those techniques which are based upon s o l u b i l i t y differences are
q u i t e similar t o t k e o l d b w r y procedure.
251.
1
Slide 1 9
Determination of Connective Tissue i n Muscle
--
Muscle Sample
I
Weak NaOH o r
1
Solution
of
Muscle Proteins
-
Strong s a l t
I
Res idue
(elastin)
I
Residue
Boiling Water
o r hot d i l u t e a l k a l i
1
Solution
(gelat i n )
The major d i f f i c u l t y with t h i s type of procedure i s the f a i l u r e
t o g e t clean separation of t h e collagen, e l a s t i n , and muscle proteins. The
n e u t r a l s a l t soluble and acid soluble collagen is l o s t by t h e hydroxide
extraction, and some of t h e muscle proteins may not be completely extracted
and w i l l be determined as collagen. Also, any insoluble nitrogeneous residue w i l l be determined as e l a s t i n . Kastelic (12) has given an excellent
discussion of these problems a t t h e Seventh Research Conference.
Determination of collagen by t h e hydroxyproline technique i s more
s p e c i f i c , since it i s n ' t influenced by nitrogeneous impurities. However,
only collagen can be determined by t h i s technique and. some assumption must
be made concerning the hydroxyproline content of the collagen. Also, use
of t h e Neuman-Logan procedure still requires t h e e x t r a c t i o n (and therefore
l o s s of soluble collagen) of the muscle proteins s i n c e a high l e v e l of
amino a c i d s o t h e r than hydroxyproline r e s u l t s i n a decreased. color format i o n . Recently however, three d i f f e r e n t hydroxyproline procedures have
been published which circumvent t h e need f o r p r i o r extraction of the muscle
proteins (24, 27, 40). The use of these techniques would avoid t h e loss of
soluble collagen and higher values f o r t h e collagen content of meat might
be expected. T h i s has been confirmed by t h e use o f a modification of the
Prockup-Udenfriend technique a t Wisconsin. These newer techniques appear
t o have s e v e r a l advantages over t h e old Neuman-Logan method f o r hydroxyprol i n e determination i n meat and should be preferred f o r f u t u r e work i n t h i s
area.
In summary, it appears that t h e r o l e of t h e three-dimensional
s t r u c t u r e of the connective t i s s u e components i n meat tenderness needs t o
be more thoroughly investigated. Attempts should be made t o follow changes
i n t h e three-dimensional s t r u c t u r e and s o l u b i l i t y of both t h e muscle and
t h e connective t i s s u e proteins during post-mortem aging and cooking. The
new hydroxyproline techniques o f f e r a convenient method of monitoring t h e
s o l u b i l i z a t i o n of collagen. The e f f e c t s of changes i n t h e mucoprotein
ground substance needs t o be studied and t h e molecular nature of e l a s t i n
more completely understood. Additional knowledge on these points should
aid g r e a t l y i n c l a r i f y i n g the molecular basis of meat tenderness (or t h e
l a c k of it!).
252.
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I
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(Applause )
MR. B R I S W : Thank you, Darrel. Again a t t h i s time, I would l i k e
t o have you record your questions and we'll cover them a l l a f t e r t h i s session. Now, we'll move on t o t h e next paper, Photometric Method f o r t h e
Determination of Elastin and Collagen i n b s c l e . We a r e very happy t o have
Bob Henrickson, who c e r t a i n l y needs no introduction t o give t h i s presentation.
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