RELATION
OF
AMINO
PROTEIN
COMPONENTS
AND
ACIDS TO PORK QUALITY 1
FREE
W. R. USBORNE,2 JAMES D. KEMP AND W. G. MOODY 3
University o] Ken'tucky, Lexington
ORK quality must be adequately defined,
p and
objective, quantitative methods must
be available before proper standards and
guidelines can be established and improvements can be made. A better understanding of
the fundamental relationships of muscle proteins and their components to pork quality
might contribute to a more precise and knowledgeable definition and methodology.
Hegarty et al. (1963) fractionated beef
muscle proteins and showed that sarcoplasmic
protein per total fibrillar protein nitrogen and
soluble fibrillar protein nitrogen were correlated with tenderness. Hill (1962) found more
stroma and myofibril protein nitrogen and less
sarcoplasmic protein nitrogen in beef muscle
when compared with pork and lamb muscles.
Ma et al. (1961) studied the free amino acid
composition of various beef muscles and concluded that the more tender cuts had a higher
leucine and isoleucine content. Several amino
acids have been associated with beef flavor
(Batzer et al., 1962).
The objectives of this research were (a)
to study the changes that take place from the
raw to the cooked product in the various protein nitrogen components and free amino acids
of porcine muscle and (b) to study the relationships of these nitrogen components and
free amino acids to color, firmness, texture,
marbling, flavor, juiciness, tenderness, WarnerBratzler shear values and over-all satisfaction
of the 1. dorsi muscle from five weight groups
of closely related hogs.
Materials and M e t h o d s
The animals in this experiment and the
procedures used were the same as those reported by Usborne et al. (1968). In addition,
the drippings from cooking were collected and
analyzed for free amino acids.
After cooking, the drippings were poured
into a beaker, the fat allowed to rise, and then
1 T h e investigation reported in this paper (67 5-82) is in
connection with a project of the Kentucky Agricultural Experiment Station and is published with apprnval of the Director.
2 Present address: Department of Animal Science, University
of Minnesota, St. Paul.
s The authors gratefully acknowledge the assistance of the
University of Kentucky Computing Center in analyzing data.
~90
each sample was refrigerated overnight. The
solidified fat was carefully removed and the
remainder saved for free amino acid analyses.
A 2.5-ml. sample of the drippings was diluted
to 25 ml. with distilled water and a 0.75-ml.
aliquot was used in the final analysis.
Analysis of variance and simple and partial
correlations were conducted following the
method of Steel and Torrie (1960). Partial
correlations were calculated to eliminate the
effect of liveweight differences. The treatments
were raw vs. cooked pork for the protein nitrogen components, and raw vs. cooked pork and
the drippings vs. raw plus cooked pork for the
free amino acids. Correlation coefficients for
all possible combinations of variables in this
study were presented by Usborne ( 1967 ). Only
pertinent partial correlation coefficients are
given in this paper.
Results and Discussion
Protein Nitrogen Components. All the protein components with the exception of total
nitrogen and collagen showed a significant
( P ~ . 0 1 ) change from the raw to the cooked
state, as shown in table 1. A comparison of
the raw and cooked values revealed that the
sarcoplasmic protein, non-protein, residual
connective tissue protein and soluble fibrillar
protein nitrogens decreased about 1O0%, 33 %,
43.5% and 75%, respectively, in the cooked
I. dorsi muscle. The decrease in soluble proteins
is in general agreement with work by Ginger
et al. (1954) and Griswold (1955). These decreases were compensated for by a tremendous
increase in the percent total fibrillar protein
nitrogen fraction.
Apparently the heat denatured and coagulated all of the sarcoplasmic protein, most of
the soluble fibrillar protein and some of the
residual connective tissue protein. Some of
the nonprotein nitrogen could have been volatilized, and this may have contributed to the
aroma of the cooked pork. New flavor compounds also may have been formed from the
raw components. Many of the compounds responsible for meat aroma and flavor have
591
P R O T E I N COMPONENTS AND I'ORK QUALITY
T A B L E 1. M E A N S AND S T A N D A R D D E V I A T I O N S OF P R O T E I N C O M P O N E N T S
State of muscle ~'
Raw
Cooked
Variables
Mean
S.D.
Mean
S.D.
Significance
%
%
%
%
%
%
%
12.17
22.41
9.89
14.44
9.49
46.40
6.86
1.11
2.10
0.80
3.30
11.78
0.00
6.58
8.17
2.33
78.60
7.05
1.02
0.00
0.37
3.95
0.35
3.47
0.75
.,
**
**
**
**
**
..
total nitrogen"
sarcoplasmic protein nitrogen b
non protein nitrogen b
residual connective tissue protein nitrogen b
soluble fibrillar protein nitrogen b
total fibrillar protein nitrogen b
collagen nitrogenb
2.21
2.59
0.68
Of muscle on dry weight basis.
b Of total nitrogen.
c N=20.
** P~.01.
been identified (Hornstein and Crowe, 1964;
Landmann and Batzer, 1966).
The means and standard deviations of the
quality and palatability characteristics referred to throughout this study appear in table
2. Marbling was the most variable of all the
components. Selected partial correlation coefficients for the raw pork protein nitrogen
components are shown in table 3. Percent
T A B L E 2. M E A N S AND S T A N D A R D D E V I A TIONS OF Q U A L I T Y AND P A L A T A B I L I T Y
CHARACTERISTICS
State of musCe
Raw
Variables
Quality characteristics
Lean color ~
Lean firmness b
Lean texture ~
Marb ling ~l
Cooked
Mean
S.D.
2.70
2.85
1.95
6.55
0.57
0.75
0.60
1.47
Palatab ility characteristics
Tenderness ~
Warner-Bratzler shear r
Flavor ~
Juiciness ~
Over-all satisfaction ~
Mean
S.D.
7.96
4.71
7.72
7.10
7.60
0.30
0.83
0.20
0.40
0.26
9 Color: 2 -pale, 3--uniformly grayish pink.
~*Firmness: 2--soft, 3 firm, 4 very firm.
e Texture: 1--coarse, 2--medium, 3 fine,
d Marbling: 5--small, 6--modest, 7 moderate.
e 9--like extremely; 1----dislikeextremely.
r Ng. force to shear 2.54-cm. core.
total nitrogen in the l. dorsi muscle showed
significant partial negative correlation coefficients with marbling, flavor, juiciness and
over-all satisfaction. The amount of total
nitrogen in lean meat seemed to play an important role in influencing the palatability
characteristics as exemplified by the high
negative partial correlations between the percet total nitrogen and the palatability charac-
teristics. The percent sarcoplasmic protein
nitrogen was significantly correlated with
firmness, juiciness and over-all satisfaction.
The percent soluble fibrillar protein nitrogen
of raw pork was significantly correlated with
marbling, but not with tenderness or WarnerBratzler shear values as Hegarty et al. (1963)
found with beef. Tenderness differences were
perhaps too small in this pork study to justify
a valid comparison. A significant partial correlation coefficient also was found between the
percent residual connective tissue protein nitrogen and juiciness. Protein nitrogen components
from cooked pork were not significantly correlated with quality and palatability characteristics.
If partial correlation coefficients were used
as an indication of relativity between the protein-nitrogen components and the quality and
palatability traits, the percents of total nitrogen and sarcoplasmic protein nitrogen of raw
T A B L E 3. S E L E C T E D P A R T I A L C O R R E L A T I O N
COEFFICIENTS--RAW MUSCLE PROTEIN
COMPONENTS VS. QUALITY TRAITS
Variables
P a r t i a l correl ation coefficients
Percent total nitrogen v s . m a r b l i n g
Percent total nitrogen v s . flavor
Percent total nitrogen v s . juiciness
Percent total nitrogen v s . over-all
satisfaction
Percent sacroplasmic protein nitrogen
v s . firmness
Percent sacroplasmic protein nitrogen
v s . juiciness
Percent sacroplasmic protein nitrogen
v s . over-all satisfaction
Percent soluble fibrillar protein nitrogen
v s . ma rbl i ng
Percent residual connective tissue protein
nitrogen v s . juiciness
* P<.05,
** P < . O I ,
- - . 44*
--. 51"
- - . 79**
- - . 72 **
0.58**
0,51"
0.45*
0.47*
- - . 58**
592
USBORNE, K E M P AND MOODY
TABLE 4. MEANS AND STANDARD DEVIATIONS OF FREE AMINO ACIDS
State of muscle
Raw (R)
Cooked (C)
Drippings (D)
Variables (Molar %)
Mean
S.D.
Mean
S.D.
Mean
S.D.
Serine
Glutamic acid
Valine
5.68
5.74
3.91
0.83
1.48
0.70
6.60
7.86
4.50
0.74
1.10
0.58
6.23
7.21
4.72
0.60
1.15
0.65
Isoleucine
Leucine
Tyrosine
2.21
4.27
3.39
0.39
0.65
0.86
3.42
7.82
4.35
0.38
1.15
0.75
2.97
6.27
3.25
0.39
0.55
0.66
Phenylalanine
2.17
0.45
3.69
0.41
2.64
0.47
Lysine
Arginine
Glycine
2.04
1.77
20.01
0.63
0.45
5.07
2.74
2.74
16.59
0.45
0.40
3.43
2.54
2.39
15.43
0.41
0.38
3.10
Alanine
26.19
3.46
20.87
2.10
26.34
2.69
Methionine
3.49
2.26
2.43
0.27
1.52
0.68
Proline
Aspartic acid
Threonine
Histidine
Total free amino acids
(~moles/gm. muscle)
P<.05.
~ P%.Ol.
6.77
1.62
5.38
3.94
5.92
4,97
1.40
0.98
0.99
0.90
5,00
1.34
5.86
4.18
8.41
0.88
0.51
1.10
0.73
0.97
5.53
1.45
6.43
5.07
32.50
0,63
0.52
0.58
1.52
5.77
pork were the most important of the traits
studied.
Free Amino
Acids.
Means and standard
deviations of the free amino acids are given
in table 4. There was much variation in the
individual observations for some of the free
amino acids, as indicated by the large standard
deviations. Serine, glutamic acid, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine
and arginine proportions were significantly
( P < . 0 1 ) higher in cooked than in raw pork.
The increase in these amino acids was probably
the result of protein degradation. Whether
selectivity was taking place in freeing the
amino acids could not be determined.
Glycine, alanine, and methionine ( P < . 0 i ) ;
and proline ( P < . 0 5 ) significantly decreased
in the cooked pork. I t has been shown that
heat from cooking does not destroy these
amino acids (Schweigert e t a l . , 1949, 1951).
These amino acids could have contributed to
the formation of new flavor compounds
(Batzer e t a l . , 1962; L a n d m a n n and Batzer,
1966). Also, this decrease could have been due
to their concentration remaining fairly constant, and because other free amino acids increased proportionally, these would have decreased. There was no significant difference
Significance
R vs. C**
Rvs. C**
R vs. C**
D vs. R+C **
R vs. C**
R vs. C**
R vs. C**
D vs. R+C **
R vs. C**
D vs. R-[-C *
R vs. C**
R vs. C**
R vs. C**
D vs. R+C**
R vs. C**
D vs. R+C **
R vs. C**
D vs. R+C**
Rye. C*
None
Dvs. R-[-C*
D vs. R+C**
D vs. R-t-C**
between the total free amino acids of raw and
cooked pork, although the total was higher
for the cooked product.
When the free amino acids in the drippings
were compared with those in the raw plus
cooked pork,
valine
(P~.01),
alanine
(P~.01),
threonine
(P~.05),
histidine
( P ~ . 0 1 ) , and the total free amino acids
( P ~ . 0 1 ) were significantly higher and tyrosine ( P ~ . 0 1 ) , phenylalanine ( P ~ . 0 5 ) , glycine ( P ~ . 0 1 ) , and methionine ( P ~ . 0 1 ) were
significantly lower. There can be so m a n y
variables--such as the initial level of the free
amino acids in the muscle, the rate of heating
during cooking, the moisture content of the
meat, the solubilities of the amino acids, and
the p H of the meat and drippings--which
could influence the general amino acid pattern
that it would be difficult to draw any definite
conclusions without more detailed studies.
Selected partial correlation coefficients for
the free amino acids from raw pork v s . the
quality and palatability characteristics are
given in table 5, Glutamic acid was significantly correlated with texture, flavor and
tenderness. Leucine showed a highly significant
positive correlation with tenderness. Some indication of the relation of leucine to tender-
593
PROTEIN COMPONENTS AND PORK QUALITY
TABLE 5. SELECTED PARTIAL CORRELATION
COEFFICIENTSIRAW MUSCLE FREE
AMINO ACIDS V S . QUALITY TRAITS
Variables
Glutamie acid vs. texture
Glutamic acid vs. flavor
Glutamic acid vs. tenderness
Leucine vs. tenderness
Serine vs.. tenderness
P henylalanine vs. tenderness
P~'.O5.
~ P<.OI,
Partial correlation coefficients
-- .49"
--. 52
0.44*
0.56"*
0.50*
0.51"
ness in beef has been reported by Ma e t a l .
(1961). Serine and phenylalanine also were
correlated with tenderness (P < .05).
Table 6 gives selected partial correlation coefficients for the free amino acids from cooked
pork v s . quality and palatability characteristics. The amino acids were most frequently
correlated with flavor, followed by juiciness
and over-all satisfaction. Tyrosine and g!utamic
acid were
negatively
correlated
( P < . 0 1 ) , aspartic acid and serine were negatively correlated ( P ~ . 0 5 ) , while glycine was
positively correlated ( P < . 0 5 ) with flavor.
T y r o s i n e and glutamic acid also were negatively correlated ( P ~ . 0 5 ) with juiciness and
over-all satisfaction. The high negative correlations with flavor showed that as the amounts
of these amino acids increased, flavor scores
decreased. One interpretation could be that
these amino acids contributed to new compounds during cooking. Three of the five
significantly correlated free amino acids (glutamic acid, serine and glycine) were the same
that Batzer e t a l . (1962) found in a hydrolyzate from a glycoprotein which served as a
precursor in beef flavor. Also, a Maillard-type
reaction may be occurring between certain
amino acids and carbohydrates (Hornstein
TABLE 6. SELECTED PARTIAL CORRELATION
COEFFICIENTS--COOKED MUSCLE FREE
AMINO ACIDS V S . QUALITY TRAITS
Variables
Tyrosine vs. flavor
Tyrosine vs. juiciness
Tyrosine vs. over-all satisfaction
Glutamic acid vs. flavor
Glutamic acid vs. juiciness
Glutamic acid vs. over-all satisfaction
Aspartie acid vs. flavor
Serine vs. flavor
Glycine vs. flavor
P<,05.
r P<.01.
Partial correlation coefficients
--. 57**
--. 46*
--. 60"*
--. 66**
--. 54
--. 48
--. 54*
--.46*
0.49*
and Crowe, 1964; Tart, 1966). This reaction
is believed to contribute to flavor production
in meat.
Lysine was significantly correlated with
marbling, glycine with color of muscle, and
aspartic acid with tenderness when the free
amino acids of the drippings were correlated
with the quality and palatability characteristics as shown in table 7. No apparent reasons
are available for these significant correlations.
Protein
Nitrogen
Components
vs.
Free
Amino
A c i d s . Selected partial correlation co-
efficients between the free amino acids and the
protein nitrogen components of raw pork are
given in table 8. Free threonine was significantly ( P < . 0 5 ) correlated with the percent
total nitrogen and arginine with the non-protein nitrogen. Glycine ( P < . 0 5 ) ,
valine
( P < . 0 1 ) and leucine ( P < . 0 5 ) were significantly correlated with the percent collagen
nitrogen. Glycine also was negatively correlated with the percent residual connective
tissue protein nitrogen.
TABLE 7. SELECTED PARTIAL CORRELATION
COEFFICIENTS--FREE AMINO ACIDS FROM
DRIPPINGS V S . QUALITY TRAITS
Variables
Lysine vs. marbling
Glycine vs. color of lean
Aspartic acid vs. tenderness
P~.o5.
Partial correIation coefficients
--. 46*
0.46*
0.45*
When the partial correlation coefficients of
the free amino acids v s . the protein nitrogen
components of the cooked meat were summarized, several of the amino acids were correlated with the percent collagen nitrogen of
the total nitrogen. These partial correlation
coefficients appear in table 9. Alanine was
negatively correlated (P ~ . 0 5 ) with percent
total nitrogen. Proline ( P < . 0 1 ) and glycine
( P ( . 0 5 ) were positively correlated with the
percent collagen nitrogen content. These two
amino acids alone contribute approximately
45% of the total amino acids found in collagen (White e t a l . , 1964). Leucine and
methionine ( P < . 0 5 ) and valine, isoleucine
and phenylalanine ( P < . 0 1 ) also were correlated with the percent collagen nitrogen but
in a negative respect.
The free amino acids in the drippings were
compared with the protein nitrogen components of cooked pork. Selected partial correlations are shown in table 10. Glycine was nega-
594
USBORNE, KEMP AND MOODY
T A B L E 8. S E L E C T E D P A R T I A L C O R R E L A T I O N
COEFFICIENTS--RAW MUSCLE FREE AMINO
ACIDS VS. R A W P R O T E I N C O M P O N E N T S
Variables
Threonine vs. % total nitrogen
Arginine vs. % non protein nitrogen
Glycine vs. % collagen nitrogen
Valine vs. % collagen nitrogen
Leucine vs. % collagen nitrogen
Glycine vs. % residual connective
tissue nitrogen
Partial correlation coefficients
0.44*
0.53*
0.70**
--.46"
- - . 46*
- - . 65**
* P<.05.
** P~.OI.
tively correlated ( P < . 0 5 ) with the percent
non protein nitrogen, tyrosine and isoleucine
( P < . 0 5 ) with the percent collagen nitrogen,
and alanine ( P < . 0 5 ) with the percent total
nitrogen.
Whether there is any significance in the
relation of each individual free amino acid to
the percent collagen nitrogen is questionable.
However, sew_~n of the free amino acids from
cooked pork and two from the drippings were
significantly correlated with collagen. Those
amino acids which were negatively correlated
are present in collagen in only small amounts.
It is difficult to make any definite statements
about the relation of tenderness, collagen and
the free amino acids since there was little difference in tenderness and the percent collagen
nitrogen failed to change significantly from
the raw to the cooked product. There was no
free amino acids significantly correlated with
tenderness in the cooked product even though
many were correlated with collagen. In raw
pork, leucine was the only free amino acid
which was significantly correlated with both
tenderness and collagen nitrogen content.
Concentrations of free leucine, possibly, could
be used as an iindicator of tenderness, but more
evidence is needed from a wider range of conditions.
T A B L E 9. S E L E C T E D P A R T I A L C O R R E L A T I O N
COEFFICIENTS--COOKED MUSCLE FREE
A M I N O ACIDS VS. C O O K E D P R O T E I N
COMPONENTS
Variables
Manine vs. % total nitrogen
Proline vs. % collagen nitrogen
Glycine 'vs. % collagen nitrogen
Leucine vs. % collagen nitrogen
Methionine vs. % collagen nitrogen
Valine vs. % collagen nitrogen
Isoleucine vs. % collagen nitrogen
Phenylalanine vs. % collagen nitrogen
* P~.05.
** P~.01.
Partial correlation coefficients
- - . 54*
0.60**
0.52*
--.44*
- - . 55*
- - . 56**
- - . 59"*
- - . 66**
Summary
Four closely related litters of five Hampshire barrows each were raised under the same
conditions and distributed among five slaughter weight groups so that each litter was
represented in each group. Various quality
and palatability characteristics, certain protein
nitrogen components, and 16 individual free
amino acids were determined on raw and
cooked samples of the I. dorsi muscle. The
drippings from cooking also were analyzed for
the free amino acids.
The percents of soluble fibrillar protein
nitrogen, sarcoplasmic protein nitrogen, non
protein nitrogen, and residual connective tissue
protein nitrogen decreased in the cooked
product when compared to the fresh state by
T A B L E 10. S E L E C T E D P A R T I A L C O R R E L A T I O N C O E F F I C I E N T S - - F R E E A M I N O ACIDS
F R O M D R I P P I N G S VS. C O O K E D P R O T E I N
COMPONENTS
Variables
Glycine vs. % non protein nitrogen
Tyrosine vs, % collagen nitrogen
Isoleucine vs, % collagen nitrogen
Alanine vs. % total nitrogen
Partial correlation coefficients
- - .45*
--.45*
- - . 56**
- - . 61"*
P<.05,
** P~.0I.
about 75%, 100%, 33% and 43.5% respectively. The percent total nitrogen increased
about 45.4%, and the percent collagen nitrogen remained almost constant from the raw
to the cooked state. Proportions of free glycine,
alanine, methionine and proline decreased in
the cooked product while proportions of free
serine, glutamic acid, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine and
arginine increased. When the free amino acids
in the drippings were compared with those
in the raw plus cooked muscle, valine, alanine,
threonine, histidine, and the total free amino
acids were higher and tyrosine, phenylalanine,
glycine and methionine were lower.
Significant partial correlation coeff•
were found for percent of total nitrogen of
raw pork with marbling (--.44"), flavor
(--.51"), juiciness (--.79**) and over-all
satisfaction (--.72**). Such coefficients also
were found for percent sarcoplasmic protein
nitrogen of raw pork with firmness (0.58"),
juiciness (0.51") and over-all satisfaction
(0.45*); free glutamic acid of raw pork with
flavor (--.52*) ; free glutamic acid (--.66"*),
tyrosine (--.57"*), aspartic acid (--.54*),
PROTEIN COMPONENTS AND PORK QUALITY
serine (.46*) and glycine (0.49*) of cooked
pork with flavor; free glutamic acid (0.44*),
leucine (0.56"*), serine (0.50*) and phenylalanine (0.51") of raw pork and aspartic acid
(0.45*) of the drippings with tenderness.
Significant partial correlation coefficients also
were found for free glycine (0.70**), valine
(--.46*) and leucine (--.46*) in raw pork
along with free proline (0.60"*), glycine
(0.52"), leucine (--.44), methionine (--.55"),
valine (--.56"*), isoleucine (--.59**) and
phenylalanine (--.66**) in cooked pork and
tyrosine (--.45*) and isoleucine (--.56**) in
the drippings with the percent collagen nitrogen.
Various individual free amino acids in
cooked pork were most frequently correlated
with flavor followed by juiciness and over-all
satisfaction.
Literature Cited
Batzer, O. F., A. T. Santoro and Vq. A. Landmann.
1962. Identification of some beef flavor precursors.
J. Agr. Food Chem. 10:94.
Ginger, I. D., J. P. Wachter, D. M. Doty, B. S.
Schweigert, F. J. Beard, J. C. Pierce and O. G.
Hankins. 1954. Effect of aging and cooking on the
distribution of certain amino acids and nitrogen
in beef muscle. Food Res. 19:410.
Griswold, R. M. 1955. The effect of different methods
of cooking beef round of commercial and prime
grades. II. Collagen, fat, and nitrogen content.
Food Res. 20:171.
Hegarty, G, R., L. J. Bratzler and A. M. Pearson.
595
1963. The relation of some intracellular protein
characteristics to beef muscle tenderness. J. Food
Sci. 28:525.
Hill, F. 1962. Fibre composition of tough and tender
muscles of meat animals. Nature 196:65.
Hornstein, I. and F. F. Crowe. 1964. Meat flavor-a review. J. Gas Chromatogr. 2:128.
Landmann, W. A. and O. F. Batzer. 1966. influence
of processing procedures on the chemistry of meat
flavors. J. Agr. Food Chem. 14:210.
Ma, R. M., M. B. Matlack and R. L. Hiner. 1961.
A study of the free amino acids in bovine muscle.
J. Food Sci. 26:485.
Schweigert, B. S., B. A. Bennett and B. T. Guthneck.
1951. Further studies on the amino acid composition of pork and lamb cuts. J. Biol. Chem. 190:697.
Schweigert, B. S., B. T. Guthneck, H. R. Kraybill
and D. A. Greenwood. 1949. The amino acid composition of pork and lamb cuts. J. Biol. Chem.
180:1077.
Steel, R. G. D. and J. H. Torrie. 1960. Principles and
Procedures of Statistics. McGraw-Hill Book Co.,
New York.
Tarr, H. L. A. 1966. Flavor of fesh foods, p. 190-202.
R. F. Gould (ed.). Flavor Chemistry. Am. Chem.
Soc., Washington, D. C.
Usborne, W. R. 1957. The relation of certain protein
components and free amino acids to quality of
porcine muscle from five different weights of hogs.
Ph.D. Dissertation. Univ. of Ky., Lexington.
Usborne, W. R., James D. Kemp and W. G. Moody.
1968. Effect of liveweight on quality, proximate
composition, certain protein components and free
amino acids of porcine muscle. J. Animal Sci.
27:584.
White, A., P. Handler and E. L. Smith. 1964. Principles of Biochemistry (3rd ed.). The Blakiston
Division, McGraw-Hill Book Company, New York.