242. CHARACTERIZATION AN0 OETERMINA TlON OF MUSCLE CONtdEC T I YE T I'SSUE COMPONNZN TS

The muscle components have long been linked with meat tenderness and for this reason their determination and, more re- cently, their characterization have received considerable attention from meat researchers. Already in the 1930rs, Helser, Bull, Mitchell, Hamilton, and others reported a negative correlation between the quantity of connec- tive tissue and meat tenderness. However, these early studies were handi- capped by the lack of adequate techniques for the quantitative determination of connective tissue. In 1941, I~wryand comrkera (15) published a tech- nique for and determination Based on the insolubility or" these proteins. Neuman and Logan (22) presented the first adequate method for hydroxyproline determination in 1950. Although both of these techniques are still widely used today in modified forms, several studies have shown that neither is completely satisfactory for the determination of connective tissue in meat (1, 12, 16, 39). bbre recently, the chemical makeup and the three-dimensional structure of connective tissue components have been extensively investi- gated (6). These studies have revealed mrked differences in the chemical structure of connective tissue isolated from animals of different ages. It io not unreasonable to wsume that the three-dimensional structure as well as the amounts of the connective tissue cornnents might be involved in meat tenderness. In fact, this disregard of the three-dimensional struc- ture may be responsible for some of the conflicting reports regarding the importance of connective tissue in meat tenderness.

In order to make an intelligent analysis of the problems of con- nective tissue determination and characterization, it is necessary to be familiar with its chemical composition and the chemical and physical prop- erties of its components. Connective tissue consists of three morphologi- cally distinct constituents.

his paper was written during the tenure of a Predoctoral Fellowship from the Division of General Medical Sciences, United States Rblic Health Service. 243.

Slide 1 Constituents of Connective Tissue

*--- *--- Cells ----Ground Substance Proteins 1. 1. 1, Collagen (embryonic cells),

2. Fat cells 2. Keratosulfate 2. Elastin

3. blast cells 3. A 3, I?e-ticulin 4. Ivlacrophages 4. Chondroitin sulfate B

5. Mesenchymal 5. Chondroitin sulfate C

cells 6. Heparin

(undifferentiated) 7, Reparitin’ SUI-fate -- The connective tissue cells will not be discussed since Dr. Nullins has already discussed these components, particularly the mast cella, at last year’s Reciprocal Meats Conference (20).

Although up to now the gmund aubstance has not been extensively investigated, recent studies have provided ~omeevidence on the chemical structure of the mucopolysaccharides found in the ground substance (19).

Slide 2 bbcopolysaccharides of Connective Tissue

Mucopolysaccharide Constituents Linkage Hyaluronic acid N-acetylglucosamine (1) (1 -+3) glucuronido- glucosamine

Glucuronic acid (1) (1-+4) glucosarninido- glucuronic acid Keratosulf ate N-acetylglucosamine (1) Galactose (1)

Sulfate (1) Hepa rin Glucosamine (1) Glucuronic acid (1) Sulfate (3) Heparitin sulfate Glucosamine (1) Glucuronic acid (1)

Sulfate (1) Slide 3 Mucopolysaccharides from Connective Tissue - Mucopolysaccharide Constituents Linkage Chondroitin sulfate A N-acetylgalactosamine (1-+3) glucuronido- galactoeamine Glucuronic acid (1) (1-4) galac tosaminido- glucuronic acid Sulfate (1) Chondroitin sulfate B N-acetylgalactosamine (143) iduronido- galac tosarnine L-idumnic acid (1) (1- 4) galacto saminido- iduronic acid Sulfate (1) Chondroitin sulfate C N-acetylgalactoaamine (l-+ 3) glucuronido- galactosamine Glucuronic acid (1) (144) wactosaminido- glucuronic 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 im- portance 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 dif- ferent mucopolysaccharides components have also been shown to differ among different sources and this my 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 be- havior is much more complex than this classification might indicate. 245,

Slide 4

Proteins from Connective Tissue Collagen Raqtin Reticulin 1. Banded under the 1, Not banded under the 1. Precollagen fibers 8 electron microscope EM-700 .. 2. Not stretched 1% by 2. Easily stretched 2. Highly branched 1,000 times its own weight

3. Solubilized to 3. Not affected by hot 3. Partially hydro- gelatin by hot . aqueous extraction lyzed by trypsin aqueous extraction Not hydrolyzed by 4. 4, Not hydrolyzed by 4. Contains some trypsin trypsin lipid 5. Eydrolyzed by 5. Hydrolyzed by collagenase, elastase, ficin, ficin, papain, and and papain some bacterial enzymes

6. Crystalline 6. Amorphous structure structure

Reticulin is a poorly characterized protein which greatly re- sembles collagen, although it is not as widespread. it possesses dif- ferent staining properties than collagen, contains a Also,tightly-bound, myristic acid-rich Z.ipid, ad some kinds of it appear to be resistant to collagenase. Since it is not present in large amounts, it should not be a major factor in meat tenderness.

Ivbscle is reported to contain about 1/3 as much elastin as collagen. Elastin isn't markedly affected by hot aqueous solvents and thus, it shou3.d play an important role in meat tenderness. The amino acid com- position of elastin may explain its insolubility in aqueous solvents since it contains over 90s nonpolar amino acids by weight. It is similar to collagen in its glycine and proline content, containing 27% and 13$ of these two amino acids respectively. These nonpolar amino acids together with the presence of a lipid which appears to be tightly bound to the elastin mole- cule make it quite refractory to aqueous solvents. This same unique amino acid composition may also explain the resistance of elastin to many enzymes. Very little is known about the three-dimensional structure of elastin due to the difficulty of obtaining good x-ray diffraction patterns. Recent investigation using elastase as a probe into the chemical structure of elastin may help to clarify the role of this protein in meat tenderness.

Collagen, like elastin, has an unique amino acid composition. It is the only protein known to contain hydroxylysine, containing 6-7 residues 26-6. of this amino acid per 1,000 amino acid residues. Also, it is the only protein which contains an appreciable amount of hydroxyproline although elastin contains 2$ by weight of this amino acid. Slide 5 shows the seven most abundant amino acids in 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 in collagen are nonpolar in character, the high dibasic acid and arginine content prevent collagen from being as nonpolar as elastin, Very few aromatic or sulfur containing amino acids are found in collagen and tryptophan is absent entirely. This latter fact has been used as a test for the purity of collagen preparations.

Although collagen has traditionally been thought to 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 or pH 4 citrate or acetate buffers were used in their extraction, physicochemical. studies show these fractions to be monodisperse containing protein molecules with a molecular weight of nearly 350,000. These protein molecules are thought to re resent monomers and have been named tropocollagen. They are 2800 fl long in diameter appearing in the form of a long rod in the native state.

I Electron micrograph of tropocollagen molecules magnified 100,000 X I

Rectron micrograph of heat denatured tropocollagen mlecules magnified 100,000 X A

Slide 8 I Left-handed helical peptide chains I Slide 9

Formation of the collagen I and I1 structures by the coiling of the three left-handed helical peptide chains into a right-handed super coil 1 247.

Slide 10 1 The triple helix I

Slide U. I Cross-sectional view of the triple helix I When the peptide chains are coiled into a triple helix of this type, every third position along an individual peptide chain is identical; that is, it has an identical environment. These positions may be numbered sequentially 1, 2, and 3. Because of conditions of steric hindrance, there are certain restrictions concerning the nature of the amino acids which may occupy these positions.

Slide 12 The Possible Positions of Side Chains

Collagen I Position Undef ormed De f o rtned Collagen I1 Gly only Other residues Gly only possible except Pro and Eypro I 2 Any residue Any residue Any residue Gly only Any residue Any residue except Val and Ileu

H bonding of -" Bonds to a Cannot bond the OH group neighboring within the of Hy-pro in chain within group of 3 - position 3 the group of 3 aticks out radially

The tripeptide gly-pro-hypro has been found to be the predominant product of a collagenase digest, so structure I1 is the one favored by most investigators since it can accomdate this sequence of amino acids with the least strain. Sequences of amino acids as long as 24 residues which con- tain no hydroxyproline and only small amounts of proline have also been found in these collagenase digests. Therefore, it has been proposed that the amino acid composition of the tropocollagen niolecule is not uniform along its length, but instead there exist areas of higli proline, glycine, alanine, and hydroxyproline content where every third amino acid is glycine and the sequence gly-pro-hypro predominates. These areas possess the "crystalline" triple helix structure and are attacked by collegenase. The narrow specificity of collagenase requires that every third amino acid be glycine, although alanine may be substituted for glycine with some decrease in the rate of hydrolysis. 248.

Slide 13 Specificity of Collagenase

I I( - Pro - R~ + R~ - ~ro Pm : Proline or hydroxyproline R1 = Any amino acid % = Glycine or alanine

Between these ordered crystalline areas, there are areas high in glutamic and aspartic acid. These areas don't possess the triple helix structure but are in the form of a more extended chain having a greater amount of disorder. Another structural feature of the tropocollagen mole- cule is the presence of a "tail" at either end as the result of the projec- tion of one of the three peptide chains past the other two at the end of the molecule. This tail is high in arginine, is hydrolyzed by trypsin, and is responsible for the aggregation of tropocpllagen to fom collagen fibers,

Investigators were puzzla for some tinie by the mechanism whereby these tropocollagen molecules 2800 A long aggregated to form collagen fibers with a 700 A periodic cross-banded structure. The discovery of two new types of collagen, the "fibrous long spacing" and the "segment long spacing" led to the solution of this problem.

Slide 14 1 Normal collagen under the electron microscopel

. Fibrous long spacing collagen under the electron microscope I

Slide 16

~~ 1 Segment long-spacing collagen under the electron microscope I

Slide 17

Aggregation of Tropocollagen I Normal collagen is formed by tropocollagen molecules aggregating head to tail successive chains overlapping by 1/4 of their length to give a 700 8 period. Fibrous long-spacing collagen is formed by tropocollagen molecules aggregating head to tail with alternate ch ins arranged in an antiparallel fashion with no overlap to give a 2800 It period. Segment long- spacing collagen is formed by the tropocollagen molecules aggregating side to side but not linearly. 249.

The interaction of the aggregated tropocollagen molecules with elastin and the mucoprotein ground substance forms the material we know as connective tissue.

The methods used to study these connective tissue components may be broadly classified into those used for the study of the three-dimensional structure and those used for the quantitative determination of these com- ponents. Many different physicochemical techniques are available for the study of three-dimensional structure but most of them require the isolation of the substance under study in a pure and unaltered state. Obtaining pure and undegraded connective tissue components from muscle tissue is obviously a very difficult task and for this reason, many of these techniques have not yet been used to study the muscle connective tissue components.

Slide 18

Methods for Three-dimensional Structure

1. X-ray diffraction 2. Melting curves

3. Gelation

4. Electron microscope

5. Spectroscopy

6. Optical rotation

7. Dielectric dispersion

8. Collagenase and other enzymes

This slide presents only a few of the many different methods available for the study of three-dimensional structure.

The use of x-ray diffraction in the study of the triple helical structure of collagen has already been mentioned. Since Dr. Krimm has dis- cussed this technique in some detail, nothing more will be said about it now. By melting curves, I am referring to either one of two kinds of' studies. The temperature of a solution of tmpocollagen molecules may be gradually raised and changes in the viscosity and optical rotation of the solution followed as a function of temperature, or collagen fibers may be substituted in place of the tropocollagen molecules and the temperature of the liquid in which the fibers are immersed then slowly raised until the fiber contracts to 1/3 of its original length. This temperature of con- tracture is known as the shrinkage temperature, T,, and is quite character- istic of a particular collagen sample. Both of these approaches may give some indication of the degree and strength of cross linking in the collagen molecules or fibers . 250.

Gelation studies have been discussed in a number of recent studies by Veis (32, 33, 34, 35, 36, 37). He has extracted gelatin from collagen samples by heating at 60' C. for 1 to 2 hours in pH G buffers. The gelatins obtained can be separated into several fairly homogeneous fractions of dif- fering molecular weight. The relative proportions of these fractions may again provide some infomation concerning cross linking.

Bob Caesens has already discussed some aspects of the use of the electron microscope in the study of protein structure. Its use to obtain a visual representation of the molecule is an obvious one.

S2ectroscopy includes the use of infrared, ultraviolet and nuclear magnetic resonance spectra as well as various modifications of these tech- niques. nectron spin resonance has also been found to be a useful tool for studying the formation of free radicals during enzyme reactions. These techniques, particularly the infrared and nuclear magnetic resonance spectra, provide a valuable tool for the study of the types of bonds and chemical groups which are present in a molecule as well as the orientation of these groups.

Optical rotation is a useful method for studying changes in the helical content of a molecule. It is particularly valuable for followfng changes in molecular structure due to the application of various treatments.

Dielectric dispersion is a tool which has not been extensively used in the study of protein stru-cture. It is defined as the variation of the dielectric constant with the frequency of the electric field and may give some information about the rotational freedom in the proteih molecule.

The use of collagenase and other enzymes may provide us with in- formation regarding the amino acid sequence of a protein as well as informa- tion on its three-d.imensiona1 structure. The kinetics of enzymic digestion may be markedly altered by cross linking, helical coiling, or any other phenomena which may render susceptible bonds in the protein less accessible to the enzyme (2, 9, 23, 31, 38). At lisconsin, we have recently kmbarked upon a study using melting curves, gelation, and collagenase digestion to study connective tissue sam- ples from anfmals of different ages. These techniques have revealed signifi- cant differences among connective tissue samples isolated from the different age group.

The methods used for the quantitative determination of collagen and elastin may be classified into two different groups depending on whetner they are based upon solubility differences or upon hydroxypnline determination. (I am here confining the discussion to the chemical deter- mination of collagen and elastin since the subsequent paper will present the various aspects of the histological determination of these components in detail). Those techniques which are based upon solubility differences are quite similar to tke old bwry procedure. 251.

Slide 19 -- 1 Determination of Connective Tissue in Muscle

I Muscle Sample

Weak NaOH or Strong salt 1 I Solution Residue of Boiling Water Muscle Proteins or hot dilute alkali I -1 Res idue Solution (elastin) (gelatin) - The major difficulty with this type of procedure is the failure to get clean separation of the collagen, elastin, and muscle proteins. The neutral salt soluble and acid soluble collagen is lost by the hydroxide extraction, and some of the muscle proteins may not be completely extracted and will be determined as collagen. Also, any insoluble nitrogeneous resi- due will be determined as elastin. Kastelic (12) has given an excellent discussion of these problems at the Seventh Research Conference.

Determination of collagen by the hydroxyproline technique is more specific, since it isn't influenced by nitrogeneous impurities. However, only collagen can be determined by this technique and. some assumption must be made concerning the hydroxyproline content of the collagen. Also, use of the Neuman-Logan procedure still requires the extraction (and therefore loss of soluble collagen) of the muscle proteins since a high level of amino acids other than hydroxyproline results in a decreased. color forma- tion. Recently however, three different hydroxyproline procedures have been published which circumvent the need for prior extraction of the muscle proteins (24, 27, 40). The use of these techniques would avoid the loss of soluble collagen and higher values for the collagen content of meat might be expected. This has been confirmed by the use of a modification of the Prockup-Udenfriend technique at Wisconsin. These newer techniques appear to have several advantages over the old Neuman-Logan method for hydroxypro- line determination in meat and should be preferred for future work in this area.

In summary, it appears that the role of the three-dimensional structure of the connective tissue components in meat tenderness needs to be more thoroughly investigated. Attempts should be made to follow changes in the three-dimensional structure and solubility of both the muscle and the connective tissue proteins during post-mortem aging and cooking. The new hydroxyproline techniques offer a convenient method of monitoring the solubilization of collagen. The effects of changes in the mucoprotein ground substance needs to be studied and the molecular nature of elastin more completely understood. Additional knowledge on these points should aid greatly in clarifying the molecular basis of meat tenderness (or the lack of it!). 252.

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(Applause )

MR. BRISW: Thank you, Darrel. Again at this time, I would like to have you record your questions and we'll cover them all after this ses- sion. Now, we'll move on to the next paper, Photometric Method for the Determination of Elastin and Collagen in bscle. We are very happy to have Bob Henrickson, who certainly needs no introduction to give this presenta- tion.

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