Comp. Biochem. PhysioL, 1975, VoL 51B, pp. 197 to 200. Pergamon Press. Printed in Great Britain
AMINO ACID COMPOSITION OF SKELETAL MUSCLE OF DOMESTIC BUFFALO (BOS BUBALUS L.)--II. FRACTIONATION IN THREE PROTEIN FRACTIONS AND STUDIES OF THEIR AMINO ACID PATTERN GIOVANNI COLONNA,z GAETANO IRACE,1 Cmo B ~ , 1 LUIGI M r ~ n u a AND FRANCESCO SALVATOREa 1 Istituto di Chimica Biologic.a, la Facolt~ di Medicina e Chirurgia, Via Costantinopoli 16, 80138 Napoli; a La Cattedra di Chimica Biologica, 2a Facolt~ di Medicina e Chirurgia, Via Sergio Pansini 5, 80131 Napoli; and a Istituto di Zoocultura, Facolt~ di Medicina Veterinaria, Via Veterinaria 1, 80137 Napoli, Universi~ degii Studi di Napoli, Napoli, Italy (Received 2 May 1974)
Abstraet--1. The skeletal muscle of Bos bubalus L. has been fractionated into three different protein fractions: sarcoplasmic, myofibrillar and insoluble. 2. The amino acid composition of these fractions has been determined. The content of essential amino acids in the various fractions has been found to be very high in decreasing order from the sarcoplasmic to the myofibrillar and insoluble fraction. 3. The "chemical score" followed by the indication of the limiting amino acid has been calculated to be 74 (lleu), 71 (Val) and 65 (Phe), respectively, for the sareoplasmic, myofibrillar and insoluble fraction. 4. The high nutritional value of meat fractions from the domestic buffalo from the Mediterranean area is supported by experimental data.
INTRODUCTION
IN A search for the availability o f new meat sources (Cutinelli-Ambesi et al., 1975) in the southern part o f Italy, it appeared o f interest to study the amino acid composition o f various protein fractious from the skeletal muscle of Boa buba/us L. The longissimus dorsi, which is a valuable piece o f red muscle used as a meat source, has been chosen for this analysis. D a t a are presented in this paper concerning typical protein fractious, which vary for their solubility in salt solution at different concentrations (Helander, 1957; Harrington et al., 1961; Ivanov, 1967). These fractions appeared to be useful material for a detailed study o f the meat from the buffalo, since even macroscopically this meat shows several differences in colour and structure within the bovine species (Mantovani, 1961). The data presented in this paper show: (i) separation of the skeletal muscle in three different protein fractious; (i/) their amino acid composition; and (ii/) the high nutritional value of buffalo meat. MATERIALS AND lVl~-WHODS
Fractionation procedure
The protein fractions from skeletal muscle were prepared essentially a~ording to the methods of Perry (1953)
and Perry and Zidowo (1959), but with several modifications. Frozen longissimus dorsi from male Italian water buffalo (Bos buba/us L.), 11 months old, weighing about 300 kg, was used in this study. This meat is dark red in colour with thick red fibers; as far as the morphology is concerned, the fibers show less nuclei and a large part with typical structural and contractile properties of the sarcoplasm (Mantovani, 1961). The muscle, excised immediately post mortem, was cleared of visible fat and connective tissue and kept frozen at -40°C until the start of the fractionation procedure. Frozen muscle tissue (510 g) was thawed overnight at +4°C, sawn into small pieces and placed in 5 vol. of 39 mM sodium borate buffer, pHT.1, containing 25mM KCI. The volume calculation is based on the original muscle weight. All the following steps were done at + 4°C unless otherwise stated. Muscle pieces added to the buffer solution were blended in an Omni Mixer homogenizer (Sorvall) for 2 m in. The homogeaate was adjusted to pH 7.1 by adding 0.1 N NaOH solution. The homogenate was then centrifuged at 2000 g for 15 min in a Sorvall RC2 B refrigerated centrifuge. The supernatant, filtered through glass wool in order to remove particles of fat that might interfere with sampling, was submitted to ceatrifugafion at 30,000 g for 60 min. The clear superna~nt containing the sarcoplasmic fraction (SAR) was decanted and stored. The sediment obtained from the first 2000 g centrifugation was placed in an Omni Mixer and homogenized for 2 min with 5 vol. of the original K ( ~ - 4 ~ t e buffer. The suspension was centrifuged at 600 g for 15 min. The
197
198
GIOVANNICOt.ONNAet al.
supernatant containing residual sarcoplasmic proteins was discarded. The precipitate together with the viscous upper layer of sediment was suspended in 5 eel. of KCIborate buffer and centrifuged for 3 rain at 400 g. The precipitate consisting of the insoluble fraction (INS) was collected and saved for subsequent analyses. The supematant containing mainly myofibrils was subjected to a further centrifugation for 15 min at 600 g. The sediment was resuspended in 5 eel. of the original buffer and centrifuged for 3 rain at 400 g. The precipitate was discarded and the supernatant subjected to a further centrifugation for 15rain at 600g. The sediment obtained from the above centrifugation was collected and resuspended in 5 vol. of the original buffer and then centrifuged at 400 g for 3 rain. The supernatant was centrifuged at 600 g for 15 rain and the sediment collected and used as the myofibrillar fraction (MYO). The overall fractionation procedure is summarized in Fig. I. MUSCLE TISSUE -homr#nffl in 5 yd. ~fler* -centriluge at Z~O9 for 15rnin
1
1
~[OIMENT -honlogeniz~in 5 vel ~ffer ~ -¢entriftJge• ~Og for IS rain.
SUPERNATANT -centrifuge
~
at 3Ohm g
SUPEII~T~ 1"~
min.
SEDIMENT Idiscard)
l
SUP£RNATANT (disr~ffl)
1
SEDIMENT I -resus~endedin 5 vd I~fler ~ -centrilu~ at 4039fer 3 rain.
l
I
S[OIW~t',T
SUPERNATANT -¢entrifug., at for 15 rain. I $UPERNATANT Idiscard)
SEDIM[NT I -resuscend~in 5 ~ . ~ f f e r ' -centrifuge el/£Og for 3 mir~. SOPER~ATA~qT I -centrilugeat 600g for IS rain.
S~I~NT (|isrJrd) I S[DIMI~T | - sulp~ld~
SUP~TA~T (0im.'d)
I
,
)
SUPIgINATANT I -ce~rHu~ it ¢~Og
in
S vd.buffer
-centrifugn a1400gfor 3rain
SEDIMENT 4discMdI
f0¢ 15 rain. f
SUPIglNMANT |discard)
SEDIMENT
Ng
Fig. 1. Fractionation of skeletal muscle proteins in three main fractions: SAIL sarcoplasmic proteins (myoglobin, myoalbumin, globulin X, enzyme soluble proteins, etc.); INS, insoluble proteins (collagen, elasfin, reticulin, etc.); MYO, myofibt~ar proteins (myosin, actin, tropomysin, etc.). * Buffer: 0.39 M borate-boric acid buffer, pH 7.1, containing 0.25 M KCI.
Protein determination
The fractions were assayed for protein content by the method of Lowry et al. (1951) using bovine serum
albumin as standard. Protein analysis of insoluble fraction (INS) was performed after enzymatic digestion with pepsin. An amount of sample containing roughly 2 g of protein was digested with 11200 (w/w) pepsin at pH 2.0 and 37°C. Lowry et aL's method was then used only when the sample appeared clear (usually 1-2 hr). Amino acid analysis
Quantitative amino acid analyses were performed on an automated amino acid analyzer (Beckman Amino Acid Analyzer, Model 120C, with a Beckman integrator, Model 125) (Spackman et aL, 1958). For each analysis, volumes of solution containing 1.0 mg of protein were dialyzed against several changes of distilled water. The solutions were lyophilized, the residues redissolved in 5 ml of 6 N HCI containing 0.01 M phenol to protect the tyrosine residues against oxidation and then placed in Pyrex vials. The vials were sealed under vacuum and kept at 105°C for 18, 36 and 72 hr, respectively. Duplicate hydrolyses were always made. After hydrolysis the samples were dried under vacuum in a desiccator containing NaOH pellets. The values for threonine and serine were estimated by linear extrapolation to zero time of hydrolysis to correct for disruption during hydrolysis. The values for valine and isoleu¢ine were determined by linear extrapolation to infinite time because of the slow rate of hydrolysis of certain peptide bonds involving these amino acids. RESULTS AND DISCUSSION The method for preparation of the three different protein fractions from the skeletal muscle was derived from that of Perry (1953) and Perry & Zidowo (1959). Following the described modifications it has been possible to obtain sarcoplasmic, myofibrillar and insoluble fractions from the same sample by a rapid procedure. The analysis of a m i n o acid composition in the three different protein fractions (SAIL, MYO and INS, as specified under Materials and Methods) trom longissimus dorsi from B. bubalus L. is reported in Table 1. The data reported are average values from the hydrolyses carried out for different periods of time as indicated under Materials and Methods, with the exception indicated in the footnote of Table 1. The difference in the amino acid composition of the three fractions studied is clearly apparent from the data. The S A R fraction is essentially formed by proteins soluble in very dilute salt solutions, which include sareoplasmic proteins as enzyme proteins and chromoproteins including myoglobin. The high percentage of this latter makes the amino acid composition of this fraction similar to that of myoglobin which is itself extracted from the same source (Balestrieri et al., 1973). The MYO fraction contains sarcoplasmic proteins which axe insoluble in water but soluble in high salt solutions, represented by the myofibrillar contractile proteins.
Amino acid composition of buffalo muscle protein fractions The I N S fraction is formed by all the other stromatic and interstitial proteins, which are completely insoluble in aqueous solutions. The high content of glycine, alanin¢ and proline in the insoluble fraction is worthy of note; this is most likely due to the high content of proteins from the connective tissue, which are very rich in these amino acids. In the same Table 1. Amino acid composition of protein fractions from the skeletal muscle of domestic buffalo Amino acids (g/100 g of protein)* Amino acid Lys His Arg Asp Thrt Sert Glu Pro Gly Ala Val~ Met Ileum: Leu Tyr Phe
SAR
MYO
INS
11"7 7"8 4-9 9"0 5"2 4.2 11-9 3.0 5-0 6.1 7-4 3.9 5.9 12.1 4.3 5.8
10"2 2"5 6"7 10"4 5"8 4"8 18"4 2.7 3.9 7.9 5.2 3.8 6.1 10.8 3"5 4-8
8"0 1"8
7"6 9-8 4-2 3"1 17.5 7.5 12.4 9"1 5.7 2.8 5.4 7.9 3"7 4.1
* Values are the average of two determinations for 18, 36 and 72 hr hydrolysates. "f Values obtained by linear extrapolation to zero time of hydrolysis. :~Values obtained by linear extrapolation to infinite time of hydrolysis. fraction there is a low content o f lysine, which is also in agreement with the data reported for collagen-like proteins. Particularly striking are the differences for glutamic acid and histidine, in the three different fractions. As far as the nutritional value of the proteins examined is concerned,, it is well known that it is related to the content of the essential amino acids. In Table 2 data are given concerning the content of essential amino acids (except tryptophan), and the Table 2. Essential amino acids, "chemical score" and limiting amino acid for three protein fractions of domestic buffalo's skeletal muscle Essential
amino acid* Chemical Fraction (g/100 g of protein) score SAR MYO INS
52-0 46.7 37"2
* Except for tryptophan.
74 71 65
Limiting
amino acid Ileu Val Phe
199
"chemical score", according to Mitchell & Block (1946) for the three ~ o n s is also ~ t e d . According .to'this ~ the most valuable fraction for the nutritional aspect is represented by the soluble fntetion (SAR fraction). I n fact, the "chemical score" is the expression o f the correlation between amino acid composition and the nutritional value of proteins. Therefore, not only the total content of essential amino acids is rather high in S A R fraction, as compared with other proteins of high nutritional value (F.A.O., 1970), but also each essential amino acid is represented in an elevated amount. In the other two fractions the content of essential amino acids is equal to 89~o (MYO) and 75~0 (INS) with respect to the soluble fraction. Also the "chemical score" shows a similar behavior, thus decreasing in the same order from the S A R to the MYO and INS fractions. The relatively high content in lysine in all three fractions as compared with the other proteins (see Table 1 in Mitchell & Block, 1946) is also worthy of note. In fact, the importance for the growth of a high lysine content in the diet has been clearly demonstrated by an extensive literature (see Scrimshaw & Altschul, 1971). Acknowledgements--This work has been supported by a C.N.R. special program (grant No. 70.01554/32/ 17.14.3) on "New protein sources". REFERENCES B~J~Tnnnu C., COLONNA G. & IRACE G. (1973) The skeletal muscle myogiobin of the water buffalo (Bos buba/us L.) Comp. Biochem. Physiol. 46B, 666-672. ~-AMBeSl L., BOCCHINI V., B,*,Lm'rlm~ C. & SALVATOgE F. (1975) Amino acid composition of skeletal muscle of domestic buffalo (Bos bubalus L.)-I. Comparative studies and nutritional value. Comp. Biochem. Physiol. 51B, 193-195. F.A.O. (1970) Amino acid content of foods and biological data on proteins. Nutrition Division, F.A.O., Roma, Italy. ~GTON W. F. & yON HII"Pr_LP. H. (1961) The structure of collagen and gelatin. Adv. Prot. Chem. 16, 1138.
HE~'qWR E. (1957) On quantitative muscle protein determination: sarcoplasm and myofibril protein content of normal and atrophic skeletal muscles. Acta phystoL scand. 41, Suppl. 41, 1-99. IVANOV I. L (1967) Organic substances of the striated muscle. Syrup. biol. Hung. 8, 89-124. LowRY O. H., ROmmROUGHN. J., F ~ A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagen t. J. biol. Chem. 193, 265-275. M~'TOV,t~,rt G. (1961) Ispezione su alimentl di origine animale. Unione Tipografica Editoriale Torinese, Torino, Italia. MrrcHrJ~ H. H. & BLOCK R. G. (1946) Some relationships between the amino acid contents of proteins and their nutritive values for the rat. J. biol. Chem. 163, 599-620.
200
GzovJ~a~NlCOLO~A et al.
P~ggg S. V. (1953) The protein components of the isolated myoflbril. Biochem.J. 55, 114-122. P~g~tYS. V. & ZYvowo M. (1959) The nature of the extra protein fraction from myofibrils of striated muscle. B/ochem. J. 71, 220-228. SCmmHAW N. S. & ALTSCHULA. M. (1971) Amino acid fortification ofprotein foods. M.LT. Press, Cambridge, Massachusetts.
SrACaCM~ D. H., STEm W. H. & MOON S. (1958) Automarie recording apparatus for use in the chromatography of amino acid. Analyt. Chem. 30, 1190-1206. Key Word lndex--Bos bubalus L.; amino acid composition; skeletal muscle; domestic buffalo; buffalo; protein fractionation.
AMINO ACID COMPOSITION OF SKELETAL MUSCLE OF DOMESTIC BUFFALO (BOS BUBALUS L.)--II. FRACTIONATION IN THREE PROTEIN FRACTIONS AND STUDIES OF THEIR AMINO ACID PATTERN GIOVANNI COLONNA,z GAETANO IRACE,1 Cmo B ~ , 1 LUIGI M r ~ n u a AND FRANCESCO SALVATOREa 1 Istituto di Chimica Biologic.a, la Facolt~ di Medicina e Chirurgia, Via Costantinopoli 16, 80138 Napoli; a La Cattedra di Chimica Biologica, 2a Facolt~ di Medicina e Chirurgia, Via Sergio Pansini 5, 80131 Napoli; and a Istituto di Zoocultura, Facolt~ di Medicina Veterinaria, Via Veterinaria 1, 80137 Napoli, Universi~ degii Studi di Napoli, Napoli, Italy (Received 2 May 1974)
Abstraet--1. The skeletal muscle of Bos bubalus L. has been fractionated into three different protein fractions: sarcoplasmic, myofibrillar and insoluble. 2. The amino acid composition of these fractions has been determined. The content of essential amino acids in the various fractions has been found to be very high in decreasing order from the sarcoplasmic to the myofibrillar and insoluble fraction. 3. The "chemical score" followed by the indication of the limiting amino acid has been calculated to be 74 (lleu), 71 (Val) and 65 (Phe), respectively, for the sareoplasmic, myofibrillar and insoluble fraction. 4. The high nutritional value of meat fractions from the domestic buffalo from the Mediterranean area is supported by experimental data.
INTRODUCTION
IN A search for the availability o f new meat sources (Cutinelli-Ambesi et al., 1975) in the southern part o f Italy, it appeared o f interest to study the amino acid composition o f various protein fractious from the skeletal muscle of Boa buba/us L. The longissimus dorsi, which is a valuable piece o f red muscle used as a meat source, has been chosen for this analysis. D a t a are presented in this paper concerning typical protein fractious, which vary for their solubility in salt solution at different concentrations (Helander, 1957; Harrington et al., 1961; Ivanov, 1967). These fractions appeared to be useful material for a detailed study o f the meat from the buffalo, since even macroscopically this meat shows several differences in colour and structure within the bovine species (Mantovani, 1961). The data presented in this paper show: (i) separation of the skeletal muscle in three different protein fractious; (i/) their amino acid composition; and (ii/) the high nutritional value of buffalo meat. MATERIALS AND lVl~-WHODS
Fractionation procedure
The protein fractions from skeletal muscle were prepared essentially a~ording to the methods of Perry (1953)
and Perry and Zidowo (1959), but with several modifications. Frozen longissimus dorsi from male Italian water buffalo (Bos buba/us L.), 11 months old, weighing about 300 kg, was used in this study. This meat is dark red in colour with thick red fibers; as far as the morphology is concerned, the fibers show less nuclei and a large part with typical structural and contractile properties of the sarcoplasm (Mantovani, 1961). The muscle, excised immediately post mortem, was cleared of visible fat and connective tissue and kept frozen at -40°C until the start of the fractionation procedure. Frozen muscle tissue (510 g) was thawed overnight at +4°C, sawn into small pieces and placed in 5 vol. of 39 mM sodium borate buffer, pHT.1, containing 25mM KCI. The volume calculation is based on the original muscle weight. All the following steps were done at + 4°C unless otherwise stated. Muscle pieces added to the buffer solution were blended in an Omni Mixer homogenizer (Sorvall) for 2 m in. The homogeaate was adjusted to pH 7.1 by adding 0.1 N NaOH solution. The homogenate was then centrifuged at 2000 g for 15 min in a Sorvall RC2 B refrigerated centrifuge. The supernatant, filtered through glass wool in order to remove particles of fat that might interfere with sampling, was submitted to ceatrifugafion at 30,000 g for 60 min. The clear superna~nt containing the sarcoplasmic fraction (SAR) was decanted and stored. The sediment obtained from the first 2000 g centrifugation was placed in an Omni Mixer and homogenized for 2 min with 5 vol. of the original K ( ~ - 4 ~ t e buffer. The suspension was centrifuged at 600 g for 15 min. The
197
198
GIOVANNICOt.ONNAet al.
supernatant containing residual sarcoplasmic proteins was discarded. The precipitate together with the viscous upper layer of sediment was suspended in 5 eel. of KCIborate buffer and centrifuged for 3 rain at 400 g. The precipitate consisting of the insoluble fraction (INS) was collected and saved for subsequent analyses. The supematant containing mainly myofibrils was subjected to a further centrifugation for 15 min at 600 g. The sediment was resuspended in 5 eel. of the original buffer and centrifuged for 3 rain at 400 g. The precipitate was discarded and the supernatant subjected to a further centrifugation for 15rain at 600g. The sediment obtained from the above centrifugation was collected and resuspended in 5 vol. of the original buffer and then centrifuged at 400 g for 3 rain. The supernatant was centrifuged at 600 g for 15 rain and the sediment collected and used as the myofibrillar fraction (MYO). The overall fractionation procedure is summarized in Fig. I. MUSCLE TISSUE -homr#nffl in 5 yd. ~fler* -centriluge at Z~O9 for 15rnin
1
1
~[OIMENT -honlogeniz~in 5 vel ~ffer ~ -¢entriftJge• ~Og for IS rain.
SUPERNATANT -centrifuge
~
at 3Ohm g
SUPEII~T~ 1"~
min.
SEDIMENT Idiscard)
l
SUP£RNATANT (disr~ffl)
1
SEDIMENT I -resus~endedin 5 vd I~fler ~ -centrilu~ at 4039fer 3 rain.
l
I
S[OIW~t',T
SUPERNATANT -¢entrifug., at for 15 rain. I $UPERNATANT Idiscard)
SEDIM[NT I -resuscend~in 5 ~ . ~ f f e r ' -centrifuge el/£Og for 3 mir~. SOPER~ATA~qT I -centrilugeat 600g for IS rain.
S~I~NT (|isrJrd) I S[DIMI~T | - sulp~ld~
SUP~TA~T (0im.'d)
I
,
)
SUPIgINATANT I -ce~rHu~ it ¢~Og
in
S vd.buffer
-centrifugn a1400gfor 3rain
SEDIMENT 4discMdI
f0¢ 15 rain. f
SUPIglNMANT |discard)
SEDIMENT
Ng
Fig. 1. Fractionation of skeletal muscle proteins in three main fractions: SAIL sarcoplasmic proteins (myoglobin, myoalbumin, globulin X, enzyme soluble proteins, etc.); INS, insoluble proteins (collagen, elasfin, reticulin, etc.); MYO, myofibt~ar proteins (myosin, actin, tropomysin, etc.). * Buffer: 0.39 M borate-boric acid buffer, pH 7.1, containing 0.25 M KCI.
Protein determination
The fractions were assayed for protein content by the method of Lowry et al. (1951) using bovine serum
albumin as standard. Protein analysis of insoluble fraction (INS) was performed after enzymatic digestion with pepsin. An amount of sample containing roughly 2 g of protein was digested with 11200 (w/w) pepsin at pH 2.0 and 37°C. Lowry et aL's method was then used only when the sample appeared clear (usually 1-2 hr). Amino acid analysis
Quantitative amino acid analyses were performed on an automated amino acid analyzer (Beckman Amino Acid Analyzer, Model 120C, with a Beckman integrator, Model 125) (Spackman et aL, 1958). For each analysis, volumes of solution containing 1.0 mg of protein were dialyzed against several changes of distilled water. The solutions were lyophilized, the residues redissolved in 5 ml of 6 N HCI containing 0.01 M phenol to protect the tyrosine residues against oxidation and then placed in Pyrex vials. The vials were sealed under vacuum and kept at 105°C for 18, 36 and 72 hr, respectively. Duplicate hydrolyses were always made. After hydrolysis the samples were dried under vacuum in a desiccator containing NaOH pellets. The values for threonine and serine were estimated by linear extrapolation to zero time of hydrolysis to correct for disruption during hydrolysis. The values for valine and isoleu¢ine were determined by linear extrapolation to infinite time because of the slow rate of hydrolysis of certain peptide bonds involving these amino acids. RESULTS AND DISCUSSION The method for preparation of the three different protein fractions from the skeletal muscle was derived from that of Perry (1953) and Perry & Zidowo (1959). Following the described modifications it has been possible to obtain sarcoplasmic, myofibrillar and insoluble fractions from the same sample by a rapid procedure. The analysis of a m i n o acid composition in the three different protein fractions (SAIL, MYO and INS, as specified under Materials and Methods) trom longissimus dorsi from B. bubalus L. is reported in Table 1. The data reported are average values from the hydrolyses carried out for different periods of time as indicated under Materials and Methods, with the exception indicated in the footnote of Table 1. The difference in the amino acid composition of the three fractions studied is clearly apparent from the data. The S A R fraction is essentially formed by proteins soluble in very dilute salt solutions, which include sareoplasmic proteins as enzyme proteins and chromoproteins including myoglobin. The high percentage of this latter makes the amino acid composition of this fraction similar to that of myoglobin which is itself extracted from the same source (Balestrieri et al., 1973). The MYO fraction contains sarcoplasmic proteins which axe insoluble in water but soluble in high salt solutions, represented by the myofibrillar contractile proteins.
Amino acid composition of buffalo muscle protein fractions The I N S fraction is formed by all the other stromatic and interstitial proteins, which are completely insoluble in aqueous solutions. The high content of glycine, alanin¢ and proline in the insoluble fraction is worthy of note; this is most likely due to the high content of proteins from the connective tissue, which are very rich in these amino acids. In the same Table 1. Amino acid composition of protein fractions from the skeletal muscle of domestic buffalo Amino acids (g/100 g of protein)* Amino acid Lys His Arg Asp Thrt Sert Glu Pro Gly Ala Val~ Met Ileum: Leu Tyr Phe
SAR
MYO
INS
11"7 7"8 4-9 9"0 5"2 4.2 11-9 3.0 5-0 6.1 7-4 3.9 5.9 12.1 4.3 5.8
10"2 2"5 6"7 10"4 5"8 4"8 18"4 2.7 3.9 7.9 5.2 3.8 6.1 10.8 3"5 4-8
8"0 1"8
7"6 9-8 4-2 3"1 17.5 7.5 12.4 9"1 5.7 2.8 5.4 7.9 3"7 4.1
* Values are the average of two determinations for 18, 36 and 72 hr hydrolysates. "f Values obtained by linear extrapolation to zero time of hydrolysis. :~Values obtained by linear extrapolation to infinite time of hydrolysis. fraction there is a low content o f lysine, which is also in agreement with the data reported for collagen-like proteins. Particularly striking are the differences for glutamic acid and histidine, in the three different fractions. As far as the nutritional value of the proteins examined is concerned,, it is well known that it is related to the content of the essential amino acids. In Table 2 data are given concerning the content of essential amino acids (except tryptophan), and the Table 2. Essential amino acids, "chemical score" and limiting amino acid for three protein fractions of domestic buffalo's skeletal muscle Essential
amino acid* Chemical Fraction (g/100 g of protein) score SAR MYO INS
52-0 46.7 37"2
* Except for tryptophan.
74 71 65
Limiting
amino acid Ileu Val Phe
199
"chemical score", according to Mitchell & Block (1946) for the three ~ o n s is also ~ t e d . According .to'this ~ the most valuable fraction for the nutritional aspect is represented by the soluble fntetion (SAR fraction). I n fact, the "chemical score" is the expression o f the correlation between amino acid composition and the nutritional value of proteins. Therefore, not only the total content of essential amino acids is rather high in S A R fraction, as compared with other proteins of high nutritional value (F.A.O., 1970), but also each essential amino acid is represented in an elevated amount. In the other two fractions the content of essential amino acids is equal to 89~o (MYO) and 75~0 (INS) with respect to the soluble fraction. Also the "chemical score" shows a similar behavior, thus decreasing in the same order from the S A R to the MYO and INS fractions. The relatively high content in lysine in all three fractions as compared with the other proteins (see Table 1 in Mitchell & Block, 1946) is also worthy of note. In fact, the importance for the growth of a high lysine content in the diet has been clearly demonstrated by an extensive literature (see Scrimshaw & Altschul, 1971). Acknowledgements--This work has been supported by a C.N.R. special program (grant No. 70.01554/32/ 17.14.3) on "New protein sources". REFERENCES B~J~Tnnnu C., COLONNA G. & IRACE G. (1973) The skeletal muscle myogiobin of the water buffalo (Bos buba/us L.) Comp. Biochem. Physiol. 46B, 666-672. ~-AMBeSl L., BOCCHINI V., B,*,Lm'rlm~ C. & SALVATOgE F. (1975) Amino acid composition of skeletal muscle of domestic buffalo (Bos bubalus L.)-I. Comparative studies and nutritional value. Comp. Biochem. Physiol. 51B, 193-195. F.A.O. (1970) Amino acid content of foods and biological data on proteins. Nutrition Division, F.A.O., Roma, Italy. ~GTON W. F. & yON HII"Pr_LP. H. (1961) The structure of collagen and gelatin. Adv. Prot. Chem. 16, 1138.
HE~'qWR E. (1957) On quantitative muscle protein determination: sarcoplasm and myofibril protein content of normal and atrophic skeletal muscles. Acta phystoL scand. 41, Suppl. 41, 1-99. IVANOV I. L (1967) Organic substances of the striated muscle. Syrup. biol. Hung. 8, 89-124. LowRY O. H., ROmmROUGHN. J., F ~ A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagen t. J. biol. Chem. 193, 265-275. M~'TOV,t~,rt G. (1961) Ispezione su alimentl di origine animale. Unione Tipografica Editoriale Torinese, Torino, Italia. MrrcHrJ~ H. H. & BLOCK R. G. (1946) Some relationships between the amino acid contents of proteins and their nutritive values for the rat. J. biol. Chem. 163, 599-620.
200
GzovJ~a~NlCOLO~A et al.
P~ggg S. V. (1953) The protein components of the isolated myoflbril. Biochem.J. 55, 114-122. P~g~tYS. V. & ZYvowo M. (1959) The nature of the extra protein fraction from myofibrils of striated muscle. B/ochem. J. 71, 220-228. SCmmHAW N. S. & ALTSCHULA. M. (1971) Amino acid fortification ofprotein foods. M.LT. Press, Cambridge, Massachusetts.
SrACaCM~ D. H., STEm W. H. & MOON S. (1958) Automarie recording apparatus for use in the chromatography of amino acid. Analyt. Chem. 30, 1190-1206. Key Word lndex--Bos bubalus L.; amino acid composition; skeletal muscle; domestic buffalo; buffalo; protein fractionation.
