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Effect of frozen temperature on autoxidation and

aggregation of bluefin tuna myoglobin in solution

Article  in  Journal of Food Biochemistry · February 2007

DOI: 10.1111/j.1745-4514.2004.tb00060.x

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 EFFECT OF FROZEN TEMPERA= ON AUTOXIDATION
AND AGGREGATION OF BLUEFIN TUNA MYOGLOBIN
IN SOLUTION
CHAU-JEN CHOW'.3, YOSHMIRO OCHIAP and SHUGO WATABE2

'National Kaohsiung Institute of Marine Technology

NO. 142, H a i - c h ~ a nRd.
Nan-Tzu. Kaohsiung 811
Taiwm, R.O.C.

zLaboratory of Aquatic Molecular Biology and Biotechnology

Graduate School of Agricultural and Life Sciences
The University of ToRyo
Bunkyo, Tokyo 113, Japan

Received for Publication March 14,2003

Accepted for Publication July 9,2003

ABSTRACT

The effect offrozen storage temperatures in the range of -3C to -80C on the
autoxidation and aggregation of blueJin tuna oxy- and deoxy-rnyoglobin (Mb)in
solution was examined. Mb solution was kept frozen at various temperatures
afrer prefreezing by dry ice-acetone and examined for arctoxidation rate constant
and aggregation ratio. The temperature-dependency of these two parameters
showed the highest values at -1OC with both oxy or deoxy form. The autoxida-
tion rate constant of deoxyMb was higher than that of oxyMb between -6Cand
-2OC, while the reverse trend was observed at lower temperatures. These results
suggest that the structure of M b was perturbed through freezing resulting in
promotion of the autoxidation and aggregation of Mb simultaneously. These
results could explain the observation that deoxyMb in the inner portion of tuna
flesh discolorsfaster than oxyMb in the surface portion in the temperature range
of -3cand -2oc.

Corresponding author. TEL: +886-7-361-7141 ext 3607; FAX: +886-7-365-8907; EMAIL:

cjchowQmail.nkimt.edu.tw

Journal of Food Biochemisay 28 (2004) 123-134. All Righrs Reserved.

'Copyright 2004 by Food & Nutrition Press, Inc.. Trumbull, Connecticut. 123
124 C.-J. CHOW, Y.OCHIAI and S. WATABE

INTRODUCTION

Frozen storage is one of the most convenient methods for the long-term
storage of fish. Tuna is customarily stored frozen on board and after landing.
Bit0 (1965a) reported that the discoloration of tuna flesh proceeded during
frozen storage at about -2OC for 6 months, while the tuna flesh hardly
discolored when it was stored frozen in the temperature range of -35C to -78C.
Discoloration of frozen tuna flesh was considered to be independent of freezing
rate, but dependent on the storage temperature (Bito 1968). In this regard,
Hashimoto and Watabe (1983) reported that the storage temperature of -4OC
was capable of preventing the discoloration provided that the tuna samples were
very fresh.
With respect to variance in the rates of discoloration, Bit0 (1965a, b) found
that the inner portion of tuna flesh block discolored at a faster rate than the
surface portion when the sample was stored at temperatures of -3C to -2OC.
Chow (1991a) also found that, with temperatures of -2OC to -3OC after storing
for 3 to 6 months, the inner portion of tuna flesh cubes discolored at a faster
rate than the surface portion. This phenomenon was related to the partial
pressure of oxygen in the muscle of tuna flesh (Bito and Honma 1967; Matsuura
ef al. 1962).
Myoglobin (Mb) is the principal pigment in tuna flesh. Due to anaerobic
conditions, the fresh flesh contains purple red deoxymyoglobin (deoxyMb) in the
inner portion. Mb then combines with an oxygen molecule to form vivid red
oxymyoglobin (oxyMb). The oxyMb and deoxyMb change into brown metMb
during prolonged storage and processing, which results in the undesirable color
of meat products (Levieux and Levieux 1996; Morita et al. 1996). It is known
that low pH value can accelerate the autoxidation of Mb (Sano and Hashimoto
1958; Brown and Mebine 1969; Shikama and Sugawara 1978), and also affects
the structure of Mb (Pan-Sun and Solberg 1972). Chow (1991b) reported that
lower stability of Mb is associated with its higher autoxidation rate. In this
respect, high correlation coefficients between the rate constant and the free
energy at different pH values were observed for several fish species (Chow
1991b). Brown and Dolev (1963) found that the autoxidation rate of yellowfin
tuna and beef Mb increased along with lowering the temperature in the range of
-5C to -15C. Satterlee and Zachariah (1972) also reported that, the autoxidation
of chicken, pig and beef Mb increased with a decrease of temperature when the
temperature was between -12C and -19C. However, few data are available on
the autoxidation of Mb solution including tuna Mb in the lower temperature
range of -4OC to -6OC that is typically selected to store tuna commercially in
Taiwan and Japan.
Previously, we observed that, after quick freezing at -8OC for 2 h and then
thawing at 20C, tuna Mb in solution showed a higher autoxidation rate than
 MYOGLOBIN AUTOXIDATION BY FREEZING 125

unfrozen Mb (Chow e? al. 1985, 1987, 1989). MetMb formation with quick
freezing at -8OC was minimum at pH values between 5.9 to 6.5 (Chow m al.
1985, 1987). Moreover, we also found that metMb formation exceeded 80%
regardless of pH values when Mb solution was frozen slowly at -2OC for 20 h.
These results indicated that the freezing temperatures affected the autoxidation
of Mb in solution significantly during the frozen storage. Besides, the rate of
Mb autoxidation in solution was obviously faster than in tuna flesh.
The present study investigated the effect of frozen temperature on the
autoxidation and aggregation of oxy and deoxy Mb in solution.

MATERIALS A N D METHODS

Materials and Purification of Mb

Mb was prepared from the dark muscle of fresh bluefin tuna (Z%unnus
thynnus) as described previously (Chow et al. 1985; Chow 1991b). All the
operations were performed in the temperature range of 3C - 4C unless otherwise
stated. Briefly, the dark muscle was coarsely minced and extracted overnight
with 2 volumes of iced distilled water under stirring. After centrifugation at
7,500 g for 15 min and subsequent filtration through Advantec No. 5C filter
paper, the filtrate was subjected to ammonium sulfate fractionation. The
precipitate formed at 50 - 80% saturation was dissolved in a small amount of
distilled water and exhaustively dialyzed against water. The dialysate was then
chromatographed using a Sephadex G-75 column (5 x 85 cm) equilibrated with
50 mM Tris-HC1 buffer (pH 8.0). The elution pattern was monitored at 280 and
540 nm. The homogeneity of Mb fraction was checked with sodium dodecyl
sulfate-polyacrylamide electrophoresis using 12.5X tube gels according to the
method of Weber and Osborn (1969) (data not shown). The Mb obtained was
of met form as judged by scanning in the visible range using a spectrophotome-
ter, and provided for the subsequent experiments immediately.

Freezing and Storage of DeoxyMb and OXyMb Solutions

Mb solution was dialyzed exhaustively against 50 mM Na-phosphate buffer
(pH 6.0) containing 0.2 M NaC1, and was used to prepare the derivatives of
Mb. All of the procedures were carried out in an ice-water bath at OC.
DeoxyMb was prepared by adding a trace amount of sodium hydrosuifite to the
Mb solution in a flask and followed by immediately purging with N, gas for 3
min. Then, 2.5 mL each of the solution was transferred into some 5-mL plastic
tubes with stopper. OxyMb was obtained by agitating deoxyMb solution gently
in air and subsequently pipeted to plastic tubes in the same way. All of these
tubes were immediately immersed in dry ice-acetone for 5 min. After keeping
126 C.-J. CHOW, Y.OCHIAI and S. WATABE

at -80Cfor 2 h, these frozen samples were, respectively, transferred to the other

freezers with temperatures at -3, -6, -10, -20, -40, and -8OC for another 24 h
or 5 days. At due time intervals, three tubes were taken out of each lot for
measurement of metMb% and aggregation ratio of Mb.

Determination of Metmyoglobin to Total Myoglobin Ratio (MetMb%)

MetMb % measurement was carried out essentially following the method of
Sano and Hashimoto (1958) with adequate modifications (Chow ef al. 1985).
The frozen Mb was thawed in a water bath at 20C for a few min and an equal
volume of 1 M Na-phosphate buffer (pH 7.0) was added and the solution was
divided into three samples. A trace of sodium hydrosulfite was added to one
sample, followed by purging with carbon monoxide (CO) gas. The absorbance
of the first sample measured at 568 nm was designated as (AJ. The second
sample was treated with CO gas directly and the absorbance measured at 568 nm
was designated as (Ad. For the third sample, 0.05 mL of 2% potassium
ferricyanide solution was added and the absorbance measured at 568 nm was
designated as (A3). The relative amount of metMb to total Mb in the solution
was evaluated as follows:

MetMb% = [(A, - A3 / (A, - A,)] x 100 (1)

The values given for the results of metMb % are the means of triplicate
analyses with standard deviations.
The autoxidation of Mb proceeded as a first-order reaction. The autoxida-
tion rate constant was calculated following the method of Matsuura ef al. (1962)
by:

K = [2 - log( 100 - metMb%)] /h (2)

Measurement of Mb Aggregation Ratio

Thawed Mb solution was filtered through a Millipore filter (pore size, 0.45
pm). Protein concentration was estimated from the absorbance at 280 nm. The
percentage of the absorbance decrement to the unfrozen original absorbance was
calculated as Mb aggregation ratio.

RESULTS AND DISCUSSION

First, the effect of storage temperature on oxyMb was examined. The

variation of metMb% and aggregation ratio of oxyMb solution during the frozen
storage at various temperatures is shown in Fig. 1. MetMb% and aggregation
 MYOGLOBIN AUTOXIDATION BY FREEZING 127

ratio increased with the storage time. A moderate variation in both parameters
was observed in the temperature range of -3Cand -80C within the 24 h storage.
However, the metMb% increased as a first-order reaction irrespective of the
frozen storage temperature between -6Cand -4OC, and was similar to the results
obtained at temperatures above OC. The rates of autoxidation were in the order
of -lOC > -2OC > -4OC > -6C> -3Cand -8OC. Chow er al. (1988)found
that the autoxidation rate constant of Mb solution decreased by reducing the
storage temperatures in the order of OC > -3C > -6C.However, f r o m Mb
solution showed higher values of metMb% than those obtained at temperature
of OC in the manner of OC < -3C< -6C.Bit0 and Honma (1967)also reported
that the temperature range causing the maximum discoloration in tuna flesh

2o
0 i
0 a 16
24

r B

0 0 16 24
Storage time (h)

FIG. 1. CHANGES IN M e w % (A) AND AGGREGATION RATIO (B) OF BL rEFIN TUNA

OxyMb IN 50 mM Na-PHOSPHATE BUFFER @H 6.0) CONTAINING 0.2 M NaCl DURING
STORAGE AT TEMPERATURES OF -3C (O), -6C (A), -lOC (O), -2OC (m), 4OC (A)
and -8OC (0)
Aliquots of oxyMb solution were quickly frozen in dry ice-acetone, and kept at -8OC for 2 h, and
then stored at those temperatures. After being thawed in a water bath at 2OC, those solutions were
measured for metMb%and aggregation ratio.
128 C.-J. CHOW,Y.OCHIAI and S. WATABE

was about -3C to -4C in the surface and about -6C to -7C in the inner part. The
results obtained in this study showed that the highest value of discoloration rate
in Mb solution was recognized at -lOC. This might be due to the presence of
0.2 M NaCl in the assay mixture.
Except at -8OC, the aggregation ratio of Mb solutions stored at temperatures
of -3C, -1OC, -2OC and -4OC increased consistently until 7 h, after which it
remained constant. The aggregation of Mb stored at -6C increased gradually
during the storage. The intensity was in the order of -6C, -1OC, and -2OC >
-4OC > -3C > -8OC. Chow ef al. (1985,1987) found that freezing and thawing
accelerated the aggregation of tuna Mb even if the Mb was quickly frozen
at -8OC for 2 h or slowly frozen at -2OC for 20 h. They suggested that freezing
and thawing promoted the conformational perturbation of Mb, especially in the
nonhelical region (Chow et al. 1989). Chen and Chow (2001) also reported that
milkfish Mb underwent some structural changes in a pH-dependent manner,
resulting in a higher susceptibility for both unfolding and autoxidation.
It is generally accepted that ice crystal formation in foods becomes
maximum at temperatures between -1 and -5C (Love and Elerian 1964). In the
present experiment, the freezing point of Mb solution might have been lowered
due to the presence of 0.2 M NaC1, so that the aggregation of Mb was promoted
in the temperature range of -6C to -2OC. The presence of salt at such a
concentration shifted the maximum ice crystal formation zone below -lOC.
Although the samples were quickly frozen in dry ice-acetone before storage, the
once-formed fine ice crystals might have grown up rapidly after the frozen
samples were transferred to the storage at the higher temperatures, resulting in
the acceleration of both Mb autoxidation and aggregation. The autoxidation and
aggregation of Mb in solution occurred quickly between -6C and -4OC within
one day of storage. It showed much faster rate of oxidation and denaturation
than Mb in the flesh. It might be related to the absence of any cryoprotectants
in Mb solution. Cryoprotectants, such as other water soluble proteins,
saccharides, and lipids in the flesh, might have prevented Mb denaturation
during frozen storage.
Secondly, the effect of storage temperature on deoxyMb was examined.
When deoxyMb solution was stored at the respective temperatures of -3C
to -8OC, similar changes of metMbZ and aggregation ratio were observed (Fig.
2). It demonstrated that both deoxy- and oxy-Mb behaved similarly at the
instants of freezing and during the subsequent frozen storage irrespective of the
presence of oxygen. The ferrous iron atom in deoxyMb can directly change into
the ferric form in metMb without going through the oxygenated ferrous form in
oxyMb. Such a result might explain that the inner portion of tuna flesh block
darkened during frozen storage at temperatures between -6C to -2OC, in spite
of anaerobic conditions in the flesh. The autoxidation rates were in the order of
-lOC > -2OC > -4OC > -6C > -3C > -8OC. The tendency in deoxyMb form
 MYOGLOBIN AUTOXIDATION BY FREEZING 129

was quite similar to oxyMb forms. On the other hand, the pattern of aggrega-
tion of deoxyMb were also similar to those of oxyMb except at -4OC, where the
ratio increased gradually during the storage. However, an explanation for this
observation remains to be elucidated.

3
0 0 a 16 24

0 8 16 24
Storage time (h)

FIG. 2. CHANGES IN MetMb% (A) AND AGGREGATION RATIO (B) OF BLUEFIN TUNA
Deoxyhfb IN 50 mM Na-PHOSPHATEBUFFER @H 6.0) CONTAINING 0.2 M NaCl DURING
THE STORAGES AT TEMPERATURES OF -3C (o),-6C (A), -lOC (a), -2OC (m), 4 C ( A )
and -8oc( 0 )
Refer to the legend in Fig. 1 for the experimental conditions in detail.

MetMb formation ratio of oxyMb and deoxyMb during storage at -3Cand

-8OC for 5 days was compared as shown in Table 1. Regardless of Mb forms
and storage temperatures, metMb96 increased along with the prolonged storage.
MetMbPrb of oxyMb remained almost constant at around 92 to 94% after 2-day
storage, while that of deoxyMb stored at -8OC increased with a lower rate than
that at -3C and those of oxyMb. It is likely that suppressing of deoxyMb
130 C.-J. CHOW, Y. OCHIAI and S. WATABE

autoxidation in tuna flesh at temperatures of -40 to -6OC could effectively inhibit

the discoloration of tuna during frozen storage.

TABLE 1.
MetMb FORMATION RATIO OF BLUEFIN TUNA OxyMb AND DeoxyMb IN
50 mh4 Na- PHOSPHATE BUFFER (pH 6.0) CONTAINING 0.2 M NaCl
DURING THE STORAGE AT -3 AND -8OC*

(%I
Temperature Storage time (day)
(C) 0 1 2 3 5
OxyMb -3 36.5*4.9 71.551.0 91.6*4.6 - 93.8*4.0
-80 36.5*4.9 78.0jz5.5 91.8+0.6 - 94.2*0.2
DeoxyMb -3 23.8*3.8 61.5*9.0 68.3*5.0 77.5*4.4 90.8*3.2
-80 23.8k3.8 38.9*5.9 36.5zk4.7 - 57.8*5.8
~

*A11 the samples were fiozen in dry ice-acetone, and kept at -80C for 2 h, and then
stored at indicated temperatures.

The temperature-dependency of the autoxidation rate constant calculated

from Eq. (2) and the aggregation ratio obtained from Mb solutions stored at
various temperatures for 3 h are shown in Fig. 3 and 4, respectively. Both the
autoxidation rate constant and aggregation ratio had the highest value at -lOC
irrespective of the myoglobin form. Temperatures for the highest aggregation
ratio were in good agreement with those found for autoxidation. Bit0 and Honma
(1967) reported that the temperatures corresponding to the maximum discolor-
ation of tuna flesh was in the range of about -3C to -4C at the surface and about
-6C to -7C in the inner part, respectively. Bit0 (1965a) also reported that the
tuna flesh hardly discolored when stored frozen in a temperature range from
-35C to -78C. Hashimoto and Watabe (1983) revealed that the lower the storage
temperature, the slower the discoloration rate, when the tuna flesh was stored
at -2OC, -4OC, -6OC and -8OC. The temperature-dependency as shown in Fig.
3 could explain the phenomenon that the discoloration of tuna flesh during the
storage at the temperatures range of -6C to -4OC was faster than at -3C or -8OC.
Moreover, Chow et ai. (1985, 1987) found that freezing and thawing not only
accelerated the autoxidation rate of tuna Mb, but also its aggregation, even if the
Mb solution was prefrozen with dry ice-acetone and then kept frozen at -8OC for
2 h or at -2OC for 20 h. Chow ef al. (1989) and Chow (1991b) also demon-
strated that the higher autoxidation rate was related to the lower stability of Mb.
The results obtained in the present study strongly suggest that the freezing
 MYOGLOBIN AUTOXIDATION BY FREEZING 131

perturbed the structure of Mb and promoted the autoxidation and aggregation of

Mb simultaneously. The aggregation ratio of oxyMb was higher than that in
deoxyMb irrespective of storage temperatures. It is possible that protein
oxidation was responsible for the aggregation during the storage.

-80 60 40 -20 0
Storage temperature (C)

FIG. 3. TEMPERATURE-DEPENDENCY OF AUTOXIDATION RATE CONSTANT OF

BLUEFIN TUNA OxyMb ( 0 ) AND DeoxyMb ( 0 )IN 50 mM Na-PHOSPHATE
BUFFER @H 6.0) CONTAINING 0.2 M NaCl
Aliquots of Mb solutions were stored at indicated temperatures after being frozen in
dry ice-acetone and kept at -8OC for 2 h

60 r

-80 -60 40 -20 0

Storage temperature (C)

FIG. 4. TEMPERATURE-DEPENDENCY OF AGGREGATION RATIO OF BLUEFIN TUNA

OxyMb ( 0 ) AND DeoxyMb ( 0 )IN 50 mhf Na-PHOSPHATE BUFFER @H 6.0)
CONTAINING 0.2 M NaCl
Aliquots of Mb solutions were stored at indicated temperatures after being frozen in
dry ice-acetone and kept at -80C for 2 h. Aggregation rntio was determined on the
initial stage of frozen samples stored at indicated temperatures for 3 h.
132 C.-J. CHOW. Y.OCHIAI and S. WATABE

The autoxidation rate constants of deoxyMb were higher than those of

oxyMb at temperature range of -6C to -2W, while the reverse tendency was
observed at the lower storage temperatures. As described above, the discolor-
ation of frozen tuna flesh block proceeds more quickly in the inner portion than
in the surface portion at -5C to -2OC (Bito 1965a, b; Chow 1991a). The
contrary observation that the inner portion discolored with a slower rate than the
surface portion during storage at lower temperature range of -35C to -78C was
also reported (Bito 1965a; Chow 1991a). Although this phenomenon has been
accounted for by a lower partial pressure of oxygen in the inner portion than in
the surface portion (Bito and Honma 1967; Matsuura et al. 1962), the present
results with bluefin tuna oxyMb and deoxyMb agree very well with the
observation found with tuna flesh block. Further studies using Mb from other
species are in progress.

CONCLUSIONS

The autoxidation rate of both oxy- and deoxy-Mb in solution associated with
freezing and thawing was in good accordance with the aggregation ratio of Mb,
regardless of the frozen storage temperatures. Both the autoxidation rate and
aggregation ratio showed the highest values at -lOC. The autoxidation rate
constants of deoxyMb were higher than those of oxyMb in the temperature range
of -6C and -2OC, while the reverse trend was observed at temperatures between
-4OC and -8OC.

ACKNOWLEDGMENTS

The research funding was defrayed in part by a Grant-in-Aid from the

Ministry of Education, Culture, Sports, Science and Technology, Japan.

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