Quantification of NADH- b5 Reductase (Metmyoglobin-Reducing ) in Bovine Skeletal Muscle by an Immunoblotting Assay

Keizo ARIHARA, Makoto ITOH and Yo KONDO

School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada-shi 034

(Received May 8, 1996)

Abstract Quantitative determination method of NADH-cytochrome b5 reductase (me- tmyoglobin-reducing enzyme) in bovine skeletal muscle was developed by combining SDS-PAGE and immunoblotting techniques. Recoveries of the enzyme by this assay ranged from 90.0 to 94.4%. The content of NADH-cytochrome b5 reductase in bovine skeletal muscle was estimated

to be 13.8±2.6(9.0-18.2)μg/g tissue (n=19). Also, a quantitative assay for determining cyto- chrome b5 (an electron transfer mediator for metmyoglobin reduction) was similarly established and cytochrome b5 content in bovine skeletal muscle was estimated to be 59.0±20.9μg/g tissue. The quantitative data of NADH-cytochrome b5 reductase in this study supported our previous hypothesis that the enzyme plays a critical role in preventing metmyoglobin accumulation in muscles. Anim. Sci. Technol. (Jpn.) 68 (1): 29-33, 1997 Key words: NADH-cytochrome b5 reductase, Metmyoglobin-reducing enzyme, Bovine skeletal muscle, Immunoblotting, Quantitative assay

Myoglobin is responsible for storage muscles, we have demonstrated the signifi- and transport in living muscles14). However, it cance of NADH-cytochrome b5 reductase is easily oxidized to metmyoglobin, resulting in (NADH: cytochrome b5 oxidoreductase, EC 1.6. the impairment of physiological functions of 2.2.) for preventing metmyoglobin accumula- . Also, myoglobin plays an impor- tion. Until recently, this enzyme in muscle tant role in post mortem change of was also called metmyoglobin reductase2,3,11). color2,8,13), an important sensory property We have proposed a new pathway that NADH affecting consumer acceptability, because meat -cytochrome b5 reductase reduces metmyo - color is dependent on the amount and the to myoglobin via either OM cytochrome chemical state of myoglobin. Desirable b or cytochrome b5 as an electron transfer cherry red color of fresh meat turns brown due mediator2,5,6). This pathway would contribute to the accumulation of metmyoglobin during to keep the chemical state of the of myo- storage. Because of these reasons, preventing globin in ferrous state in maintaining normal metmyoglobin formation is critical in keeping physiological functions of myoglobin in normal physiological functions of muscle and muscles. Therefore, this pathway would play in keeping fresh meat color. a very important role. In a series of our studies2-7) concerning Thus, it is desirable to establish a reliable metmyoglobin-reducing enzyme systems in method to determine NADH-cytochrome b5

Anim. Sci. Technol. (Jpn.) 68 (1): 29-33 29 1997 ARIHARA, ITOH and KONDO reductase content in order to make a thorough b5, reductase and cytochrome b5 previously investigation in proving the pathway. This raised in rabbits6) were used in this study, enzyme is present as a tightly membrane- Antibodies in the serum were purified by bound form in most tissues (e.g. liver, brain, affinity chromatography on CNBr-Sepharose 4 placenta, lung, muscle)15,16), and its activity is B coupled to the purified bovine NADH-cyto- relatively unstable and inactivated during ex- chrome b5 reductase or cytochrome b5 accord- traction procedures3). Since it seems likely ing to the manufacturer's protocols (Pharmacia that the concentration of endogenous interme- Fine Chemicals, Uppsala, Sweden). The diate acceptor (cytochrome b5 or OM cyto- specifity of antibodies was confirmed by an chrome b) is not saturated in those tissues, immunoblotting technique. differences in its concentration could be a SDS-PAGE and Immunoblotting: cause of the differences in the activity of the Determination of NADH-cytochrome b5 enzyme at equal enzyme concentrations. reductase and cytochrome b5 was carried out as Also, physiological intermediate acceptors for follows. SDS-PAGE was performed according the assay of enzyme activity are not available to the method of Laemmli10), using 12.5% ac- commercially. Because of these reasons, it has rylamide separating gel. Muscle samples (0.1 been difficult to examine the exact activity and g) were minced with scissors and dispersed in quantity of the enzyme, in muscles. In this Laemmli's sample buffer (1.9ml) with Potter's study, we determined the content of NADH- homogenizer for 1min before heating (100℃, 3 cytochrome b5 reductase and cytochrome b5 in min). The homogenate was centrifuged at bovine skeletal muscle by immunoblotting 5,000×g for 5min and its supernatant (10μl) techniques. was used for SDS-PAGE. Following the fractionation by SDS-PAGE, were Materials and Methods transferred from the gel to PVDF membranes Bovine skeletal muscle: (Clearblot P-membrane, Atto Co., Tokyo), as Bovine thigh muscle (biceps femoris) used for outlined by Kyhse-Andersen9), using a home establishing a quantitative method was ob- made horizontal electroblotting apparatus. tained immediately after slaughter with the Blotting was carried out with 25mM Tris-192 assistance of the laboratory of veterinary pa- mM glycine-20%(v/v) methanol buffer (pH8.3) thology staff, Kitasato University. The at 100mA for 60min. After electroblotting , muscle was stored at -20℃ until use. For the blots on the membrane were immunostained determination of NADH-cytochrome b5 using the primary antibody (anti-NADH-cyto- reductase content in beef, fresh thigh muscle chrome b5 reductase or anti-cytochrome b5, ap- samples were purchased from a local super- proximately 1μg IgG/ml) described above and market. an Bio-Rad Immun-Blot Assay Kit (Bio-Rad NADH-cytochrome b5 reductase and cyto- Laboratories, Richmond, CA, USA; secondary chrome b5: antibody, horseradish peroxidase-labelled goat NADH-cytochrome b5 reductase and cyto- anti-rabbit IgG; development reagent , 4- chrome b5 for immunization described below chloro-1-naphthol). All immunostainning and standard solutions for the assay were pre- procedures were performed according to the pared from bovine erythrocytes according to manufacturer's protocols . Pre-stained SDS- the procedures described in previous PAGE standards and SDS-PAGE low-molecu- papers3,4,6). lar-weight standards (Bio-Rad) were used to Antibodies: monitor the efficiency of electrophoretic trans - Antisera against bovine NADH-cytochrome fer of the enzyme and to determine the molecu -

30 Muscle Metmyoglobin-Reducing Enzyme lar weight of the enzyme, respectively. tration. From the standard curve shown in Absorbance and area of immunostained bands Fig. 1, the assay for this enzyme was quantita- on the membrane was measured by reflection tive over the entire range examined (4-128ng). mode at 570nm with a Shimadzu (Kyoto, As shown in Table 1, the recoveries of the com- Japan) chromato-scanner CS-930. ponent added to bovine skeletal muscle homo- genate demonstrated that the quantification Results and Discussion procedure of the present study is highly relia- After SDS-PAGE and immunoblotting, ble and gives satisfactory values. The deter- NADH-cytochrome b5 reductase in bovine skel- mination method for cytochrome b5 was also etal muscle was well recognized as a single established similarly with that for NADH-cy- band on the immunostained membrane in ac- tochrome b5 reductase. These results strongly cordance with the results obtained in the previ- indicate that the present quantitative method ous paper6). Thus, the immunological detec- based on the immunoblotting procedure is ap- tion method of the present study was proved to plicable for the determination of NADH-cyto- be highly specific for the enzyme. The molec- ular weight value (35,000) of NADH-cyto- Table 1. Recoveries of NADH-cytochrome b5 chrome b5 reductase, based on the position of reductase added to bovine skeletal muscle the immunostained band, was identical to that homogenates of a previous paper6). The optimum heating period and SDS concentration for the prepara- tion of SDS-PAGE samples were 3min and 2%, respectively. Quantification of the amount of the enzyme present in muscles was made by preparing a 1Purified enzyme perparation was added to 10μl of standard calibration curve by plotting the ab- sorbance of the purified NADH-cytochrome b5 muscle homogenate sample for SDS-PAGE. 2Average of three replicates . reductase preparation versus concen-

Fig. 1. Calibration curve for standard NADH-cytochrome b5 reductase solution. A, calibration curve drawn from the peak area of densitogram (B); B, densitogram of the membrane immunostained by anti-NADH-cytochrome b5 reductase antibodies.

31 ARIHARA, ITOH and KONDO

Table 2. Contents of NADH-cytochrome b5 reductase and cytochome b5 in bovine skeletal muscle determined by the immunoblotting procedure

1 Average of three replicates of each sample was used. chrome b5 reductase and cytochrome b5 content Acknowledgements in muscle. Averages of the contents of NADH-cyto- This work was supported in part by a Grant chrome b5 reductase and cytochrome b5 (elec- -in-Aid for Scientific Research (No. 02760165) tron-transfer mediator for metmyoglobin re- from the Ministry of Education, Science and duction)4) in bovine skeletal muscle (thighs) Culture of Japan. were estimated to be 13.5 and 59.0μg/g muscle, References respectively, by the present immunoblotting procedure (Table 2). 1) Antonini E, Brunori M. The derivatives of fer- In erythrocytes, it has been recognized that rous and myoglobin. In: Hemo- globin and Myoglobin and Their Reactions sufficient quantity of NADH-cytochrome b5 with Ligands. (Neuberger A, Tatum EL eds.) reductase (water-soluble form) is present in the 13-39. North-Holland Publishing Co. Amster- cells to prevent the accumulation of dam. 1971. (an oxidized form of hemoglo- ) Arihara K. 2Meat color and related (in bin)15,16). Hemoglobin and myoglobin have Japanese). Chikusan no Kenkyuu, 46: 13-20. 1992. common properties in the structure, derivative 3) Arihara K, Itch M, Kondo Y. Identification of formation and physiological functions1). The bovine skeletal muscle metmyoglobin weight ratio of NADH-cytochrome b5 reductase as an NADH-cytochrome b5 reductase: hemoglobin was calculated to be reductase. Jpn. J. Zootech. Sci., 60: 46-56. 1989. approximately 1:75,000 from those contents in 4) Arihara K, Itoh M, Kondo Y. Detection of cyto- human erythrocytes15). On the other hand, the chrome b5 in bovine skeletal muscle by elec- trophoretic immunoblotting technique. Jpn. J. ratio in bovine skeletal muscle, at least in Zootech. Sci., 60: 97-100. 1989. thigh, NADH-cytochrome b5 reductase: myo- 5) Arihara K, Indo M, Itoh M, Kondo Y. Presence globin is approximately 1:145-360 from the of cytochrome b5-like (OM cyto- calculation using the content of the enzyme of chrome b) in rat muscles as a metmyoglobin present study and the myoglobin content of reducing enzyme system component. Jpn. J. Zootech. Sci., 61: 837-842. 1990. bovine skeletal muscle (2-5mg/g tissue12)). 6) Arihara K, Cassens RG, Greaser ML, From the comparison of these values, sufficient Luchansky JB, Mozdziak PE. Localization of quantity of NADH-cytochrome b5 reductase metmyoglobin-reducing enzyme (NADH-cy- exists to prevent metmyoglobin accumulation tochrome b5 reductase) system components in in muscle. In conclusion, the data in this bovine skeletal muscle. Meat Sci., 39: 205-213. study supported our previous hypothesise2,5,-7) 1995. 7) Arihara K, Itoh M, Kondo Y. Contribution of that NADH-cytochrome b5 reductase plays a the glycolytic pathway to enzymatic critical role in reducing metmyoglobin to myo- metmyoglobin reduction in myocytes . globin in muscles. Biochem. Mol. Biol. Int., 38: 325-331 . 1996. 8) Faustman C, Cassens RG. The biological basis

32 Muscle Metmyoglobin-Reducing Enzyme

for discoloration in fresh meat: a review. J. 12) Maga JA. Pink discoloration in cooked white Muscle Foods, 1: 217-243. 1990. meat. Food Rev. Int., 10: 273-286. 1994. 9) Kyhse-Andersen J. Electroblotting of multiple 13) Renerre M. Review: Factors involved in the gels: a simple apparatus without buffer tank discoloration of beef meat. Int. J. Food Sci. for rapid transfer of proteins from poly- Technol., 25: 613-630. 1990. acrylamide to nitrocellulose. J. Biochem. 14) Wittenberg JB. Myoglobin-facilitated oxygen Biophys. Methods, 10: 203-209. 1984. diffusion-role of myoglobin in oxygen entry 10) Laemmii U. Cleavage of structural proteins into muscle. Physiol. Rev., 50: 559-636. 1970. during the assembly of the head of bacterio- 15) Yubisui T. Methemoglobin reductases in phage T4. Nature, 227: 680-685. 1970. erythrocytes (in Japanese). Seikagaku, 54: 11) Livingston DJ, Mclachlan SJ, La Mar N, Brown 1233-1254. 1982. WD. Myoglobin: cytochrome b5 interactions 16) Yubisui T, Takeshita M. Methemoglobin and the kinetic mechanism of metmyoglobin reducing systems (in Japanese). Tanpa- reductase. J. Biol. Chem., 260: 15699-15707. kushitsu Kakusan Kouso, 32: 854-865. 1987. 1985.

牛 骨 格 筋 にお け るNADH-チ トク ロ ムb5還 元 酵 素(メ トミオ グ ロ ビ ン還 元 酵 素)の 免 疫 プ ロ ッテ ィ ング法 に よ る定 量

有 原 圭 三.伊 藤 良 ・近 藤 洋

北里大学獣 医畜産学部,十 和田 市034

SDS-PAGEと 免疫 ブロ ッテ ィ ン グ法 に よ り,筋 肉 の メ ト ミオ グ ロ ビ ン還元に重 要 な 役 割 を 果 た して い る と報告 さ れ て い るNADH-チ トクロ ムb5還 元 酵 素 の定 量 法 を 確 立 した.こ の 方 法 で は,目 的 酵素 の 回収 率 は90.0か ら94.4%で あ った.牛骨 格 筋 に お け るNADH-チ トクロ ムb5還 元 酵 素 の 定量 を 行 っ た 結 果,13.8±2.6μg/g筋 肉(n=19)で あ っ た.ま た,NADH-チ トク ロムb5還 元 酵 素 が メ ト ミオ グ ロ ビ ンを還 元 す る際 に電 子 伝 達 の 中間 体 と して利 用 す るチ トク ロ ムb5につ い て も同 様 に定 量 法 を 確 立 した

(59.0±20.9μg/g筋 肉).本 研 究 に お け る牛 骨格 筋 のNADH-チ トクロ ムb5還 元酵 素 の 定 量 結 果 は,メ ト ミオ グ ロ ビ ンを還 元 す るの に十 分 な量 の 本 酵 素 が 筋 肉 に存 在 す る こ とを示 して い た.従 って,本 研 究 の デ ー タ は筋 肉 に お い てNADH-チ トクロ ムb5還 元 酵 素 が メ ト ミオ グロ ビ ン蓄 積 抑 制 に重 要 で あ る と い う これ まで の 仮 説 を支 持 す る もの で あ る. 日畜 会報,68(1):29-33,1997

日 畜会報,68(1):29-33 33 1997