Major buffering constituents in animal muscle

03~-9429/92 $5.00 + 0.00 @ 1992 Pergamon Press plc

Camp. Biochem. Physiaf. Vol. 102A, No. 1, pp. 37-41, 1992 Printed in Great Britain

MAJOR

BUFFERING

CONSTITUENTS MUSCLE

EMIKOOKUMA and Department

IN ANIMAL

HIROKI ABE

of Food and Nutritional Science, Kyoritsu Women’s University, Motohachioji, Tokyo 193, Japan

Hachioji,

(~ecej~e~ 28 August 1991) Abstract-l. Among the muscles of six fish species, three mammals, and a bird, white muscle of skipjack tuna showed the highest buffering capacity (BC) in the pH range 6.5-7.5, followed by the muscle of little-piked whale, chicken pectoralis minor, and mackerel white muscle. 2. Contribution of low-molecular weight components to the muscle BC was as high as 4896%, while the contribution of muscle proteins was below 50%. 3. Histidine-relate dipeptides and inorganic phosphate were found to be major buffering constituents in muscle. 4. The dipeptides accounted for the BC differences found between the species and muscle types.

INTRODUCTION

capacity of some animal muscles is known to be significantly high at physiological pH range (Castellini and Somero, 1981; Abe et al., 1985). This high buffering capacity serves to maintain the intracellular pH for the functions of proteins, such as enzymes needed during anaerobic burst exercise (Somero, 1981; Hochachka and Somero, 1984). In all vertebrate muscles examined hitherto, buffering capacity was typically higher in the white muscle of warm-bodied tuna and billfish and the skeletal muscle of marine mammals than in the muscle of most pelagic and deep-sea fishes and terrestrial mammals (Castellini and Somero, 1981; Abe et al., 1985). Possible buffering constituents in animal muscle were thought to be inorganic phosphate, nucieotides, organic acids, taurine, protein-bound L-histidine residues, free L-histidine, and L-histidinerelated dipeptides such as carnosine (b-alanyl+ histidine), anserine @-alanyl-n-methyl+histidine), and balenine (fi-alanyl-r-methyl-L-histidine) (Davey, 1960; Hochachka and Somero, 1984; Abe et al., 1985; Suyama et al., 1986). Of these buffer species having pK values around physiological pH, free L-histidine and anserine were predominant in the white muscle of Pacific blue marlin which showed extremely high anaerobic capability and high buffering capacity, while phosphate buffering was greater than the dipeptides in less active trout muscle (Abe et al., 1985). In the above situation, we intended to clarify the dominant buffering species in the animal muscles having widely different exercise abilities and different levels of L-histidine-related compounds. The six fish species under investigation were most active, skipjack tuna containing the highest amount of histidinerelated compounds of all vertebrate muscles thus far examined (Abe et al., 1986); less active were mackerel, rainbow trout, carp, eel, and sluggish flounder. These fishes were compared to a marine mammal (little-piked whale containing a large amount of balenine), terrestrial mammals (ox and pig), and a bird (chicken).

MATERIALS AND METHODS

Buffering

Rainbow trout (Oncorhynchur mykiss) and carp (Cy@zus curpio) were obtained from a local supplier in Tokyo and kept in a tank supplied with aerated tap water. Live Japanese eel (An,quilla japonica) and a flounder (Liopsetta o&cura), as ‘wei as fresh common mackerel @comber iuoonicusl were obtained from Hachioii Wholesale Fish Market in Tokyo. Also purchased at the-market were fresh bovine, porcine, and chicken muscles, and frozen skeletal muscle of little-piked whale (Balaenopteru ucutorostruta). Skipjack tuna (Katsuwonus peiamis) was caught off Hawaii and kept frozen below -85°C. White and red muscles of mackerel and white muscles of trout, carp, eel, and flounder were used for the experiment as soon as possible after decapitation or transportation to the lab. Frozen tuna as welt as whale muscle was used after cutting and thawing throughout the experiment. Bovine and porcine biceps femoris, porcine psoas muscle and chicken pectoralis minor were also used as soon as possible after sampling. Tissue fractionation

From each muscle tissue mentioned above, crude homogenate was prepared by homogenizing (Polytron PT-10, Kinematica, Zurich) 1 g of muscle tissue for 5 min in icecold salt solution containing 145 mM KCl, 10 mM NaCl and 5 mM iodoacetic acid. The homogenate was diluted with the salt solution and brought to 1OOml. Ten milliliters of the homogenate was centrifuged at 25,000g for 15 min at 0°C and the pellet was rehomopnized in 10 ml of the above salt solution and processed as above. The pellet thus obtained was homogenize with lOm1 of the salt solution and used for the determination of buffer capacity (Fig. 1). The supematants obtained were combined and separated with centrifugal ultra-filtration using a Centriprep 10 (Amicon, Denvers; exclusion limit, 10,000) into high molecular weight (HMW) and low molecular weight (LMW) fractions (Fig. 1). Both fractions were finally brought to lOm1 with the salt solution and used for the dete~ination of buffer capacity. For determining the denatured protein buffering capacity, sodium dodecyl sulfate (SDS) was added to the above homogenate, pellet, and HMW fractions of tuna white muscle to a final concentration of 10%. After homogenization their buffering capacities were determined. 37

EMIKOOKLJMA and

38

HIROKIABE

NUCLEOTIDES ORGANIC ACIDS INORGANIC PHOSPHATE

Fig. 1. Fractionation

scheme used to separate different muscle fractions measurement of buffering capacity.

Perchloric acid extract of each muscle was prepared according to the method of Abe (1991) and used for the determination of various compounds and buffer capacity (Fig. 1). Determinationof buffering capacity Buffering capacity was determined on all isolated fractions described above, i.e. the homogenate, supematant, pellet, HMW, and LMW fractions as well as perchloric acid extract, using a j-titrator (BETA-l; TOA Denpa, Tokyo) according to the previous method (Abe and Okuma, 1991a). All preparations (10 ml) were adjusted to pH 6.0 with 1 N HCl and titrated to pH 8.0 with 0.1 N NaOH at 20°C. Buffering capacity was calculated as micromoles of NaOH required per 1 g of the muscle to change the pH by one unit over the pH ranges of 6.0-7.0, 6.5-7.5, and 7S8.0. All the determinations were duplicated. Analyticalmethoris Histidine-related compounds, nucleotides, and organic acids were determined on the perchloric acid extracts by high performance liquid chromatography according to previous methods (Abe and Ohmama, 1987; Abe and Okuma, 1991b). Inorganic orthophosphate levels were determined on the perchloric acid extract according to Taussky and Shorr (1953). RESULTS AND

DISCUSSION

Buffering capacities of muscle homogenates Table 1 represents homogenates from

the buffering capacities of the animal muscles over the pH

Table 1. Buffering capacity of the crude homogenates of various muscles over the pH ranges of 6.0-7.0, 6.5-7.5, and 7X1-8.0 (pm01 NaOH/pHg muscle) Species Tuna

WM

Mackerel

;“M RM WM WM WM WM SM BF BF PSM PM

Trout Carp Eel Flounder Whale OX Pig Chicken

6.0-7.0

PH 6.5-7.5

7X&8.0

136 68.8 81.3 51.2 61.5 57.1 50.4 44.6 111 69.0 63.2 78.6 82.8

123 65.5 66.9 43.8 56.1 50.8 47.9 40.9 120 62.7 61.1 71.5 83.9

90.4 54.2 44.2 33.9 39.5 36. I 37.3 32.4 82.5 45.7 47.3 53.1 68.5

WM white muscle, RM red muscle, SM skeletal muscle, BF biceps femoris, PSM psoas muscle, PM pectoralis minor. For tuna and mackerel muscle, values shown are means of five muscles but N = 1 for the other muscle.

from homogenate.

BC;

ranges of 6.0-7.0, 6.5-7.5, and 7.0-8.0. Of all muscle homogenates, white muscle of skipjack tuna showed the highest buffering capacity in the pH range

of 6.0-7.0, followed by whale skeletal muscle. The white muscle also showed the highest buffer capacity in all animal muscles examined hitherto. In the pH range of 6.5-7.5, on the other hand, whale and tuna showed almost the same value, indicating the pK difference of histidine and balenine as described previously (Abe and Okuma, 1991a). Mackerel, a dark-fleshed fast pelagic swimmer, and chicken showed almost the same high buffering capacity, while the less active carp, eel, and flounder represented much lower capacities, below 50gmol NaOH/pH . g. Except for the whale muscle, buffering capacity of muscle homogenate decreased with increasing pH ranges due to the pK characteristics of histidine imidazole (Somero, 1981; Hochachka and Somero, 1984). Buffering capacity of fast-twitched white muscle was about twice as high than that of slow-twitched red muscle in both skipjack tuna and mackerel, which coincided with the previous data for Pacific blue marlin and rainbow trout (Abe et al., 1985). Thus, the buffering capacity of animal muscle was considered to be proportional to the anaerobic capability of the animals and muscle types. Almost a 3-fold difference of buffering capacities was found among the animal muscles (Table 1). These data almost coincided with those of Castellini and Somero (1981). Buflering capacities of muscle fractions Table 2 shows the buffering capacities of all isolated fractions (Fig. 1) in the pH range of 6.5-7.5. Buffering capacity of the pellet fraction which consists of contractile and connective tissue proteins was relatively low and rather similar across the muscles. The percentage relative contribution of the fraction to the homogenate buffering ranged from a low of 4% for tuna white muscle to a high of 25% for

mackerel red muscle and was inversely correlated with the homogenate buffering. This contribution was higher in red muscle than in white. The homogenate buffering, therefore, was recovered mainly in the supernatant fraction (above 75% for each muscle). The recovery of the homogenate buffering into the pellet and supernatant fractions was as high as from 80 to 115%, depending on the species and muscle types.

Major buffering constituents in Table

2. Buffering

capacity

of the

fractions

separated from @mol

Homogenate

Mackerel Trout Eel Flounder Whale OX

Chicken

WM RM WM RM WM WM WM WM SM BF BF PSM PM

123 65.5 66.9 43.8 56.1 50.8 47.9 40.9 120 62.7 61.1 77.5 83.9

(100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100)

crude

homogenate g mu&e)

(4.3) (14.2) (10.9) (25.1) (8.2) (13.0) (18.8) 112.51 ii3.3j (8.5) (7.9) (5.2) (5.7)

113 58.7 61.4 31.3 45.7 43.6 45.6 33.5 90.1 50.4 47.8 58.0 78.7

muscles

(91.9) (89.6) (91.8) (85.2) (81.5) (85.8) (95.2) (81.9) (75.1) (80.4) (78.2) (74.8) (93.8)

37.6 25.0 19.7 12.1 4.9 6.3 10.1 5.8 11.3 9.6 10.6 17.0 15.4

in

the

79.0 42.4 44.2 26.5 34.5 35.1 38.5 39. I 57.4 41.2 41.7 44.4 59.2

(30.6) (38.2) (29.4) (27.6) (8.7) (12.4) (21.1) (14.2) (9.4) (15.3) (17.3) (21.9) (18.4)

pH

range

6.5-7.5

PCA

LMW

HMW

Suoernatant 5.3 9.3 7.3 11.0 4.6 6.6 9.0 5.1 16.0 5.3 4.8 4.0 4.8

of various

(642) (64.7) (66.1) (60.5) (61.5) (69.1) (80.2) (95.6) (47.8) (65.7) (68.2) (57.3) (70.6)

80.6 (65.5) 46.3 (70.7) ND 32.5N(:7.9) 30.3 (48.5) 38.2 (79.7) 30.3 (74.1) 56.7 (47.3) 41.4 (66.0) 36.6 (59.9) 42.5 (54.8) 56.3 (67.11

Percentage ratios of buffering capacity of each fraction to that of homogenate are shown in parentheses. HMW high-molecular weight fraction, LMW low-molecular weight fraction, PCA perchloric acid, ND not determined. Refer to the legend of Table 1 for other abbreviations and N numbers.

Buffering capacity of the HMW fraction which consists of soluble proteins, on the other hand, was also low, and higher in dark-fleshed fishes such as tuna and mackerel than in the other white-fleshed fishes. This is consistent with the well known fact that dark-fleshed fishes contain much larger amounts of soluble proteins than white-fleshed fishes. The HMW fraction contributes from 38% for tuna red muscle to 9% for trout white muscle relative to the homogenate buffering (Table 2). Table 3 demonstrates the effects of sodium dodecyl sulfate (SDS) on the buffering capacities of the protein fractions, i.e. the homogenate, pellet, and HMW fractions from tuna white and red muscles. Buffering capacities of all fractions increased from 3 to 5% in the presence of SDS. This increase was rather large for the pellet fraction of both white and red muscles. These data suggested that not all of the histidine residues in the muscle native proteins can contribute to the total muscle buffering capacity. The buffering capacity of LMW fraction which consists of the muscle extractive components was very high irrespective of the species and muscle types and coincided well with that of the perchloric acid extract (Table 2). This fraction can contribute over 60% to the muscle buffering in all species except in whale where the recovery of buffering capacity into HMW and LMW fractions was as low as 76% due to the low value of HMW fraction. This reason was not correctly understood in the present experiment. The buffering capacity of LMW fraction was almost proportional to that of the homogenate, suggesting that the extractive components mainly determine the species difference of the buffering capacity in the

animal muscle (Table 1). Thus, the dominant buffering constituents in the animal muscle were considered to be low molecular weight compounds having pK at physiological pH range. Contribution

of nucleotides and organic acids

Table 4 gives the composition of nucleotides and organic acids in the white and red muscles of tuna and mackerel. The dominant nucleotide was inosine 5’-monophophate (pK 6.0) which was derived from ATP after the death of the animal. Of the eight organic acids, lactate (pK 3.86) was predominant in the white muscle of mackerel, followed by 2-oxoglutarate (pK 4.68). Large amounts of acetate (pK 4.74) and propionate (pK4.84), however, were found in red muscle. This may suggest the decomposition of acetyl- and propionyl-CoA during the sample preparation. Although these organic acids may hardly contribute to muscle buffering due to their low pK values, citric acid (pK 6.40) and succinic acid (pK 5.64) are considered to contribute to the buffering. As shown in Table 4, however, their contents in fish muscle were extremely limited. For determining the contribution of these compounds to muscle buffering capacity, mixtures containing the tissue levels (Table 4) of these nucleotides or organic acids were prepared using the authentic compounds and determined for buffering capacity. Extremely low buffering capacities below 5 prnol NaOH/g*pH were found in both nucleotide and organic acid mixtures in the pH range of 6.5-7.5 (data not shown). The data indicated that these compounds showed only a slight contribution to the muscle buffering at least at physiological pH and

Table 3. Effect of sodium dodecyl sulfate (SDS) on the buffering capacity of protein fractions skipjack white and red muscle @mol Na0HjpH.g muscle) PH 6.5-7.5

6.0-7.0 Fraction Homogenate Pellet HMW

-SDS WM RM WM RM WM RM

Refer to the legends of Tables

136 68.8 7.2 12.0 39.4 25.8

+ SDS 140 77.0 14.5 19.6 50.6 39.6

-SDS 123 65.5 5.1 9.3 37.6 25.0

1 and 2 for abbreviations.

from

7.k8.0 + SDS

-SDS

+SDS

130 71.0 9.3 15.0 49.1 34.8

90.4 54.2 4.9 8.2 29.8 21.7

III 63.3 6.8 10.7 43.7 30.6

EMIKOOKUMAand HIROKIABE

40 Table 4. Concentrations Species Tuna Mackerel

WM RM WM RM

of nucleotidcs and organic acids in the muscle of skipjack tuna and mackerel &mol/g muscle)

IMP

GMP AMP ADP

ATP

OX0

CIT

8.77 2.43 9.94 1.08

0.13 -

1.90 0.17 -

21.7 16.0

0.69 0.33

0.66 0.36 0.32 0.31

0.95 0.49 0.19 0.10

MAL SUC 1.77 3.72

3.88 3.55

LAC FUM

ACE

PRO

87.6 30.1

4.40 56.2

7.38 17.2

0.07 0.03

IMP inosine 5’-monophosphate, GMP guanosine 5’-monophosphate, AMP adenosine 5’-monophosphate, ADP adenosine diphosphate, ATP adenosine triphosphate, OX0 2-oxoglutaric acid, CIT citric acid, MAL malic acid, SUC succinic acid, LAC lactic acid, FUM fumaric acid, ACE acetic acid, PRO propionic acid. Refer to the legend of Table 1 for other abbreviations and N numbers.

physiological muscle.

levels of these compounds

in the

Contribution of histidine-related compounds andphosphate

The levels of histidine-related compounds in animal muscles varied according to the species and muscle types (Table 5). The total level of these compounds in the white muscle of skipjack tuna was highest in all species examined hitherto (Abe et al., 1986). Mackerel contains only histidine (pK 6.21) in large amount, in white muscle. In red muscle, however, histidine-related compounds constituted only one-seventh of those in the white muscle of both tuna and mackerel. The levels of these compounds in the white muscle of the other fishes almost coincided with previous data (Abe, 1983a,b; Abe et al., 1985). Whale muscle, on the other hand, contains large amount of balenine (pK 6.93) which is almost equivalent to thata reported by Suyama et al. (1977). Ox and pig muscle contain rather large amounts of carnosine (pK 7.01), while chicken muscle contains much anserine (pK 7.15). By contrast, inorganic phosphate (pK 6.88) showed rather small species differences, ranging from 30 to 60pmol/g, as seen in Table 5. A part of this phosphate, however, may be derived from the liberation from ATP and creatine phosphate after the death of the animal (Abe et al., 1985). For accessing the contribution of these histidine compounds and phosphate relative to the muscle buffering, a calibration curve of the buffering capacity of each compound was made using the various concentrations of authentic compounds (data not shown). Table 6 shows the buffer capacities calculated from the tissue concentrations of these compounds shown in Table 5 and their calibration curves. Only data in the pH range of 6.5-7.5 are shown in Table 6. The percentages of the buffering capacity of these compounds to the homogenate buffering are given in parentheses.

Table 5. Concentrations Tuna

His MeHis Car Ans Bal Pi

Buffer capacities of histidine-related compounds of tuna white and whale skeletal muscle were as high as 39 and 25%, respectively, relative to their muscle buffering. The ratio was also high for bovine, porcine, and chicken muscle, ranging from 12 to 23%, but only l-6% for the red muscle of mackerel and the white muscle of carp and flounder. The ratio of inorganic phosphate buffering to muscle buffering capacity in tuna white and whale skeletal muscle was relatively low, while in the white muscle of trout, carp, and flounder which contain only small amounts of histidine-related compounds the ratio was significantly high, ranging from 50 to 82%. This phosphate buffering, however, would be overestimated because phosphate was liberated from creatine phosphate or ATP after the death of the animal as described above. The contribution of total buffering capacities of histidine-related compounds and inorganic phosphate to LMW buffering was about 80%, except for the red muscle of mackerel and the muscle of ox and pig in which case the recovery was only 60 to 67%, suggesting the contribution of the other buffering species such as taurine in fish red muscle (Abe et al., 1985). No large difference in the phosphate buffering was found among the animal muscles examined in this experiment. Therefore, this phosphate buffering, ranging from 14 to 34/*mol NaOH/pH.g, is considered to contribute to the basic muscle buffering (about 40pmol NaOH/pH.g) together with the muscle proteins (10-43 pmol NaOH/pH.g). Thus, the large variation of muscle buffering capacity would mainly be attributable to the levels of histidinerelated compounds in animal muscle. These data and previous data (Davey, 1960; Castellini and Somero, 1981; Abe et al., 1985; Suyama et al., 1986) clearly indicated that the accumulation of histidine-related compounds in animal muscle raises the muscle buffering capacity and therefore the muscle anaerobic capability. This is realized in some animals such as tuna, billfishes, and marine mammals.

of histidine-related comoounds and inoreanic ohosohate found in animal muscles (UrnoVa muscle) Mackerel

Trout

Carp

Eel

Flounder

Whale

OX

Chicken

Pig

WM

RM

WM

RM

WM

WM

WM

WM

SM

BF

BF

PSM

PM

93.5

13.3 0.2 0.6 1.3

48.0 0.09 0.03 -

8.2 -

4.9 0.03 0.3 10.8

10.2 0.02 1.6 0.02

0.7 0.02 0.03

48.5

53.4

26.0

60.5

45.5

0.1 17.5 0.06 43.8

0.7 0.3 9.9 0.2 51.1 42.6

0.1 0.03 14.5 3.6 0.03 30.4

0.2 0.02 13.3 1.0 0.9 37.6

0.6 0.01 20. I 1.0 1.6 32.4

1.3 1.o 9.0 32.0 0.2 52.0

2.9 51.1 55.4

6&

His L-histidine, MeHis n- and r-methyl-L-histidine, Car camosine, Ans anserine, Bal balenine, Pi inorganic orthophosphate. legend of Table 1 for muscle abbreviations and N numbers.

Refer to the

41

Major buffering constituents in animal muscle Table 6. Calculated buffering capacities of hi&dine-related compounds and inorganic phosphate in animal muscles in the pH range 6.5-7.5 bmol

Tuna Mackerel Trout Carp Eel Flounder Whale OX Pig Chicken

WM RM WM RM WM WM WM WM SM BF BF PSM PM

NaOH/pH.g

muscle)

His

Ans

Car

Bal

His--Total

22.0 3.1 11.3 1.9 1.1 2.4 0.45 0.17 0.03 0.05 0.13 0.30

24.2 3.5 0.04 5.1 0.01 0.08 1.7 0.48 0.49 15.1

1.3 0.25 0.01 0.12 0.75 8.0 0.03 4.5 6.6 6.1 9.1 4.1

-

47.5 6.9 11.4 1.9 6.3 3.2 8.0 0.48 29.3 8.3 7.0 10.4 19.6

0.02 24.5 0.01 0.37 0.70 0.10

(38.6) (10.5) (17.0) (4.3) (11.2) (6.3) (16.7) (1.2) (24.4) (13.2) (11.5) (13.4) (23.4)

Pi 30.6 26.8 29.5 14.4 33.5 25.2 24.2 33.5 23.6 16.8 20.8 17.9 28.8

(24.9) (40.9) (44.1) (32.9) (59.7) (49.6) (50.5) (81.9) (19.7) (26.8) (34.0) (23.1) (34.3)

Percent relative contributions of total histidine-related compounds and phosphate to the muscle buffering capacity are shown in parentheses. Refer to the legends of Tables 1 and 5 for abbreviations and Table 1 for N numbers. Acknowledgements-This

work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. The authors are indebted to Miss N. Kobayashi and Mrs M. Nakao for their technical assistance. REFERENCES

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