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Functional molecular size of trypsin inhibitors as determined by radiation inactivation analysis
Biochimica et Biophysica Acta 914 (1987) 101-103 Elsevier BBA 30208
101
BBA Report
Functional molecular size of trypsin inhibitors as determined by radiation inactivation analysis Tamikazu K u m e and Isao Ishigaki Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Gunma (Japan) (Received 6 February 1987)
Key words: Radiation inactivation; Molecular size; Ovomucoid; Trypsin inhibitor; Domain size
The molecular sizes of trypsin inhibitors obtained by radiation inactivation were investigated in relation to the functional domain size. The molecular weights obtained were 10 200 + 700 for ovomucoid, 17 800 5:400 for ovoinhibitor and 16400 + 500 for soybean trypsin inhibitor. These values mostly agreed with the size of domain which has trypsin inhibitory activity, suggesting that the radiation inactivation analysis indicates the minimum functional domain size.
The molecular sizes of various enzymes can be measured by the radiation inactivation method [1]. The principle of the radiation inactivation method is based on the target theory that a single hit of an ionizing radiation on a macromolecule will completely destroy its biological activity. The target size of the molecule can, therefore, be estimated from the degree of inactivation [2,3]. Since the radiation inactivation method offers the distinct advantage that solubilization or purification of the macromolecule is not required [4], this method has been widely used to determine the molecular size of enzymes [5] and receptors [6,7] in situ. However, many of the results do not agree with those obtained from other procedures [1]. During a study of the effect of irradiation on chicken ovomucoid, which consists of three domains, one of which has trypsin-inhibiting activity [8,9], we found that the molecular size of ovomucoid obtained by radiation inactivation was smaller than the expected molecular weight. This Correspondence: T. Kume, Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Gunma 370-12, Japan.
paper describes the molecular size of various trypsin inhibitors, obtained by radiation inactivation, in relation to the functional domain in viva and in vitro. Ovomucoid was a generous gift of Dr. T. Matsuda of the Faculty of Agriculture, Nagoya University and had been isolated from the egg white of White Leghorn hens and purified by column chromatography [10,11]. Ovoinhibitor and soybean trypsin inhibitor were obtained from Sigma (Types 3-0 and 1-S, respectively). Lysozyme was crystallized three times from the egg white of White Leghorn hens [12]. The other enzymes, trypsin and pepsin, were obtained from Miles-Seravac (Grade 5) and ICN Pharmaceuticals (three times crystallized from hog stomach mucosa), respectively. These samples were lyophilized after dissolving in distilled water, sealed in vacua, and irradiated at room temperature approx. 25°C with 190 kCi (7.0 pBq) 6°Co slab source. The dose rate used was 1.0 M r a d / h (10 k G y / h ) as determined by Fricke dosimetry. Trypsin inhibiting activity was assayed by measuring the initial rate of increase in absorbance at 420 nm with a-N-benzoyl-p-nitroanilide, as described
0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
102
1 8O 6O
0
i
0
i
(
f
~
i
i
TABLE I MOLECULAR WEIGHT OBTAINED BY RADIATION INACTIVATION AND HPLC ANALYSIS Molecular weight (M,) was calculated from 037 dose as described in the text.
>', 40 .>_
Reported M r by
< 2O 10
0
i 10
i 20
i 30
I 40
J 50
Dose (Mrod) Fig. 1. Radiation inactivation of three trypsin inhibitors. Purified trypsin inhibitors were lyophilized, sealed in vacuo and irradiated at room temperature. The lines, O, ovomucoid; o, ovoinhibitor; zx, soybean trypsin inhibitor, represent data analyzed by the method of least squares constrained to 100% at zero dose.
by Waheed and Salahuddin [13]. tysozyme and pepsin activities were measured according to the method of Shugar [14] and Rick and Fritsch [15], respectively. H PLC analysis was carried out using a TSK-GEL G3000SW (Toyo soda, 7.7 × 600 mm) with a pre-column (7.5 x 75 ram) [16]. The samples were eluted with 0.2 M potassium phosphate buffer (pH 7.0) at a flow rate of 0.7 ml/min. Marker proteins from Oriental Yeast (glutamate dehydrogenase, 290 000; alcohol dehydrogenase, 142 000; enolase, 67 000; adenylate kinase, 32 000, and cytocrome c, 12400) were used for molecular weight determination. Three trypsin inhibitors, ovomucoid, ovoinhibitor and soybean trypsin inhibitor (Kunitz), were inactivated exponentially and the /)37 doses were 62.7, 36.0 and 39.0 Mrad, respectively (Fig. 1). From these 037 doses, molecular weight (Mr) was calculated by the following empirical equation of Kepner and Macey [5] Mr = 6.4 × 10S/D37
where D37 is the dose (Mrad) necessary to inactivate an activity to 37% of its initial level. The activities of the known molecular size enzymes, lysozyme and pepsin, also decreased exponentially and the D37 doses were 45.1 and 19.3 Mrad, respectively. Table I shows the molecular sizes calculated from these D37 doses, with reported molecular weights and the values obtained by
Ovomucoid Ovoinhibitor Soybean trypsin inhibitor Lysozyme Pepsin
Radiation inactivation
M,
HPLC D37 Mr (Mrad)
28000 49000
55 000 55000
62.7 36.0
10200 +__ 700 17800+_ 400
20000 14300 35000
19500
39.0 45.1 19.3
16400_+ 500 14200_+ 300 33200+1800
HPLC analysis. The molecular weight obtained by HPLC analysis shows that the ovomucoid used in this experiment is a dimer, whereas the other trypsin inhibitors are monomers. The molecular size of 14 200 for lysozyme and 33 200 for pepsin obtained by radiation inactivation agrees with the reported molecular weight of 14300 and 35000 [15]. One of us [17] reported earlier that the molecular weight of 152000 for glucose isomerase obtained by radiation inactivation in a dry cell agrees well with the molecular weight of 157000. These results show that the dosimetry and the obtained molecular weight by radiation inactivation are quite reliable. The molecular size of ovomucoid and of ovoinhibitor obtained by radiation inactivation were 10200 + 700 (mean _+ S.D: n = 5) and 17 800 _+ 400, respectively. These are smaller than the earlier reported molecular weights of 28000 and 49000. The molecular weight of 16400 + 500 for soybean trypsin inhibitor is close to the expected value of 20000 [18]. Ovomucoid consists of three domains, one of which shows trypsin-inhibiting activity. Thus, the molecular weight of domain with trypsin inhibiting activity is estimated to be 9 300. Ovoinhibitor, another trypsin inhibitor in egg white, consists of seven domains, four of which have trypsin inhibiting activity [8,9]. Since 1 mol ovoinhibitor inhibits 2 mol trypsin, the molecular weight of the domain with trypsin inhibiting activity is estimated to be 14000. The values obtained by radiation inactiva-
103
tion almost agree with these estimated values. Ovomucoid, ovoinhibitor and lysozyme are all components of chicken egg white. The inactivation of trypsin inhibitor and lysozyme in egg white was investigated to determine the functional size in situ. The trypsin inhibiting activity in egg white depends mainly on ovomucoid, because the chicken egg white proteins contain 11% ovomucoid and 1.5% ovoinhibitor [19]. The molecular size of the trypsin inhibitor in egg white may, therefore, represent mainly the molecular size of ovomucoid. The activities of trypsin inhibitor and lysozyme irradiated in egg white decreased exponentially and the D37 doses were 42.1 and 31.5 Mrad, respectively. The molecular size of trypsin inhibitor and lysozyme calculated from /)37 doses were 15200+500 and 2 0 3 0 0 + 6 0 0 and these values were somewhat bigger than 10200 and 14200 obtained in vitro (Table I), respectively. When pepsin in egg white was irradiated, the D37 dose was 19.0 Mrad and the molecular weight was obtained as 33 700 + 1300, which agreed with those in vitro. This shows that the addition of egg white has no effect on the radiation inactivation. Therefore, the bigger molecular sizes in egg white mean that some interaction with other components increase the target size for the radiation in situ. Ovomucoid and ovoinhibitor are glycoprotein containing 20-25% and 5-10% carbohydrates, respectivelY. If these carbohydrates do not affect the target size, as reported by Lowe and Kempner [20], the functional domain size will be estimated as somewhat smaller. Considering the reported temperature effect on target size [21], the functional size increased only 3% at 25°C as used in this experiment. From all of the above results, we conclude that the molecular size of proteins obtained by radiation inactivation indicates the functional domain size. We thank Dr. T. Matsuda of the Faculty of Agriculture, Nagoya University for a gift of purified ovomucoid and suggestion. We also appreciate discussion of Dr. H. Ito of our Institute.
References 1 Kempner, E.S. and Schlegel, W. (1979) Anal. Biochem. 92, 2-10 2 Okada, S. (1970) Radiation Biochemistry (Altman, K.I., Gerber, G.B. and Okada, S., eds.), pp. 11-17, Academic Press, New York 3 Pollard, E.C. (1951) Biophysical Science - A Study Program, pp. 273-278, John Wiley & Sons, New York 4 Harmon, J.T., Nielsen, T.B. and Kempner, E.S. (1985) Methods Enzymol. 117, 65-94 5 Kepner, G.R. and Macey, R.I. (1968) Biochim. Biophys. Acta 163, 188-203 6 Venter, J.C., Schaber, J.S., U'Prichard, D.C. and Fraser, C.M. (1983) Biochem. Biophys. Res. Commun. 116, 1070-1075. 7 Wang, N.I., Fukui, H., Matsuoka, H. and Wada, H. (19861 Biochem. Biophys. Res. Commun. 137, 593-598 8 Kato, I., Kohr, W.J. and Laskowski, M., Jr. (1978) in Regulatory Proteolytic Enzymes and their Inhibitions, l l t h FEBS Meeting (Magnusson, S., Ottesen, M., Foltman, B. Dano, K., Neurath, M., eds.), pp. 197-206, Pergamon
Press, Oxford 9 Kato, I. (1982) Protein, Nucleic Acid Enzyme 27, 1747-1756 10 Watanabe, K., Matsuda, T. and Sato, Y. (1981) Biochim. Biophys. Acta 667, 242-250 11 Matsuda, T., Watanabe, K. and Nakamura, R. (1982) Biochim. Biophys. Acta 707, 121-128 12 Kume, T., Sato, Y. and Umemoto, Y. (1973) Nippon Nogeikagaku Kaishi 47, 549-555 13 Waheed, A. and Salahuddin, A. (1975) Biochem. J. 147, 139-144 14 Shugar, D. (1952) Biochim. Biophys. Acta 8, 302-309 15 Rick, W. and Fritsch, W.P. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.V., ed.), pp. 1046-1053, Academic Press, New York 16 Kato, Y., Komiya, K., Sasaki, H. and Hashimoto, T. (1980) J. Chromatography 190, 297-303 17 Kume, T., Watanabe, H., Takehisa, M. and Sato, T. (1981) Agric. Biol. Chem. 45, 1351-1355 18 Ikenaka, T. (1982) Protein, Nucleic Acid Enzyme 27, 1738-1746 19 Parkinson, T.L. (1966) J. Sci. Food Agric 17, 101-103 20 Lowe, M.E. and Kempner, E.S. (1982) J. Biol. Chem. 257, 12478-12480 21 Beauregard, G. and Potier, M. (1985) Anal. Biochem. 150, 117-120
101
BBA Report
Functional molecular size of trypsin inhibitors as determined by radiation inactivation analysis Tamikazu K u m e and Isao Ishigaki Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Gunma (Japan) (Received 6 February 1987)
Key words: Radiation inactivation; Molecular size; Ovomucoid; Trypsin inhibitor; Domain size
The molecular sizes of trypsin inhibitors obtained by radiation inactivation were investigated in relation to the functional domain size. The molecular weights obtained were 10 200 + 700 for ovomucoid, 17 800 5:400 for ovoinhibitor and 16400 + 500 for soybean trypsin inhibitor. These values mostly agreed with the size of domain which has trypsin inhibitory activity, suggesting that the radiation inactivation analysis indicates the minimum functional domain size.
The molecular sizes of various enzymes can be measured by the radiation inactivation method [1]. The principle of the radiation inactivation method is based on the target theory that a single hit of an ionizing radiation on a macromolecule will completely destroy its biological activity. The target size of the molecule can, therefore, be estimated from the degree of inactivation [2,3]. Since the radiation inactivation method offers the distinct advantage that solubilization or purification of the macromolecule is not required [4], this method has been widely used to determine the molecular size of enzymes [5] and receptors [6,7] in situ. However, many of the results do not agree with those obtained from other procedures [1]. During a study of the effect of irradiation on chicken ovomucoid, which consists of three domains, one of which has trypsin-inhibiting activity [8,9], we found that the molecular size of ovomucoid obtained by radiation inactivation was smaller than the expected molecular weight. This Correspondence: T. Kume, Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Gunma 370-12, Japan.
paper describes the molecular size of various trypsin inhibitors, obtained by radiation inactivation, in relation to the functional domain in viva and in vitro. Ovomucoid was a generous gift of Dr. T. Matsuda of the Faculty of Agriculture, Nagoya University and had been isolated from the egg white of White Leghorn hens and purified by column chromatography [10,11]. Ovoinhibitor and soybean trypsin inhibitor were obtained from Sigma (Types 3-0 and 1-S, respectively). Lysozyme was crystallized three times from the egg white of White Leghorn hens [12]. The other enzymes, trypsin and pepsin, were obtained from Miles-Seravac (Grade 5) and ICN Pharmaceuticals (three times crystallized from hog stomach mucosa), respectively. These samples were lyophilized after dissolving in distilled water, sealed in vacua, and irradiated at room temperature approx. 25°C with 190 kCi (7.0 pBq) 6°Co slab source. The dose rate used was 1.0 M r a d / h (10 k G y / h ) as determined by Fricke dosimetry. Trypsin inhibiting activity was assayed by measuring the initial rate of increase in absorbance at 420 nm with a-N-benzoyl-p-nitroanilide, as described
0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
102
1 8O 6O
0
i
0
i
(
f
~
i
i
TABLE I MOLECULAR WEIGHT OBTAINED BY RADIATION INACTIVATION AND HPLC ANALYSIS Molecular weight (M,) was calculated from 037 dose as described in the text.
>', 40 .>_
Reported M r by
< 2O 10
0
i 10
i 20
i 30
I 40
J 50
Dose (Mrod) Fig. 1. Radiation inactivation of three trypsin inhibitors. Purified trypsin inhibitors were lyophilized, sealed in vacuo and irradiated at room temperature. The lines, O, ovomucoid; o, ovoinhibitor; zx, soybean trypsin inhibitor, represent data analyzed by the method of least squares constrained to 100% at zero dose.
by Waheed and Salahuddin [13]. tysozyme and pepsin activities were measured according to the method of Shugar [14] and Rick and Fritsch [15], respectively. H PLC analysis was carried out using a TSK-GEL G3000SW (Toyo soda, 7.7 × 600 mm) with a pre-column (7.5 x 75 ram) [16]. The samples were eluted with 0.2 M potassium phosphate buffer (pH 7.0) at a flow rate of 0.7 ml/min. Marker proteins from Oriental Yeast (glutamate dehydrogenase, 290 000; alcohol dehydrogenase, 142 000; enolase, 67 000; adenylate kinase, 32 000, and cytocrome c, 12400) were used for molecular weight determination. Three trypsin inhibitors, ovomucoid, ovoinhibitor and soybean trypsin inhibitor (Kunitz), were inactivated exponentially and the /)37 doses were 62.7, 36.0 and 39.0 Mrad, respectively (Fig. 1). From these 037 doses, molecular weight (Mr) was calculated by the following empirical equation of Kepner and Macey [5] Mr = 6.4 × 10S/D37
where D37 is the dose (Mrad) necessary to inactivate an activity to 37% of its initial level. The activities of the known molecular size enzymes, lysozyme and pepsin, also decreased exponentially and the D37 doses were 45.1 and 19.3 Mrad, respectively. Table I shows the molecular sizes calculated from these D37 doses, with reported molecular weights and the values obtained by
Ovomucoid Ovoinhibitor Soybean trypsin inhibitor Lysozyme Pepsin
Radiation inactivation
M,
HPLC D37 Mr (Mrad)
28000 49000
55 000 55000
62.7 36.0
10200 +__ 700 17800+_ 400
20000 14300 35000
19500
39.0 45.1 19.3
16400_+ 500 14200_+ 300 33200+1800
HPLC analysis. The molecular weight obtained by HPLC analysis shows that the ovomucoid used in this experiment is a dimer, whereas the other trypsin inhibitors are monomers. The molecular size of 14 200 for lysozyme and 33 200 for pepsin obtained by radiation inactivation agrees with the reported molecular weight of 14300 and 35000 [15]. One of us [17] reported earlier that the molecular weight of 152000 for glucose isomerase obtained by radiation inactivation in a dry cell agrees well with the molecular weight of 157000. These results show that the dosimetry and the obtained molecular weight by radiation inactivation are quite reliable. The molecular size of ovomucoid and of ovoinhibitor obtained by radiation inactivation were 10200 + 700 (mean _+ S.D: n = 5) and 17 800 _+ 400, respectively. These are smaller than the earlier reported molecular weights of 28000 and 49000. The molecular weight of 16400 + 500 for soybean trypsin inhibitor is close to the expected value of 20000 [18]. Ovomucoid consists of three domains, one of which shows trypsin-inhibiting activity. Thus, the molecular weight of domain with trypsin inhibiting activity is estimated to be 9 300. Ovoinhibitor, another trypsin inhibitor in egg white, consists of seven domains, four of which have trypsin inhibiting activity [8,9]. Since 1 mol ovoinhibitor inhibits 2 mol trypsin, the molecular weight of the domain with trypsin inhibiting activity is estimated to be 14000. The values obtained by radiation inactiva-
103
tion almost agree with these estimated values. Ovomucoid, ovoinhibitor and lysozyme are all components of chicken egg white. The inactivation of trypsin inhibitor and lysozyme in egg white was investigated to determine the functional size in situ. The trypsin inhibiting activity in egg white depends mainly on ovomucoid, because the chicken egg white proteins contain 11% ovomucoid and 1.5% ovoinhibitor [19]. The molecular size of the trypsin inhibitor in egg white may, therefore, represent mainly the molecular size of ovomucoid. The activities of trypsin inhibitor and lysozyme irradiated in egg white decreased exponentially and the D37 doses were 42.1 and 31.5 Mrad, respectively. The molecular size of trypsin inhibitor and lysozyme calculated from /)37 doses were 15200+500 and 2 0 3 0 0 + 6 0 0 and these values were somewhat bigger than 10200 and 14200 obtained in vitro (Table I), respectively. When pepsin in egg white was irradiated, the D37 dose was 19.0 Mrad and the molecular weight was obtained as 33 700 + 1300, which agreed with those in vitro. This shows that the addition of egg white has no effect on the radiation inactivation. Therefore, the bigger molecular sizes in egg white mean that some interaction with other components increase the target size for the radiation in situ. Ovomucoid and ovoinhibitor are glycoprotein containing 20-25% and 5-10% carbohydrates, respectivelY. If these carbohydrates do not affect the target size, as reported by Lowe and Kempner [20], the functional domain size will be estimated as somewhat smaller. Considering the reported temperature effect on target size [21], the functional size increased only 3% at 25°C as used in this experiment. From all of the above results, we conclude that the molecular size of proteins obtained by radiation inactivation indicates the functional domain size. We thank Dr. T. Matsuda of the Faculty of Agriculture, Nagoya University for a gift of purified ovomucoid and suggestion. We also appreciate discussion of Dr. H. Ito of our Institute.
References 1 Kempner, E.S. and Schlegel, W. (1979) Anal. Biochem. 92, 2-10 2 Okada, S. (1970) Radiation Biochemistry (Altman, K.I., Gerber, G.B. and Okada, S., eds.), pp. 11-17, Academic Press, New York 3 Pollard, E.C. (1951) Biophysical Science - A Study Program, pp. 273-278, John Wiley & Sons, New York 4 Harmon, J.T., Nielsen, T.B. and Kempner, E.S. (1985) Methods Enzymol. 117, 65-94 5 Kepner, G.R. and Macey, R.I. (1968) Biochim. Biophys. Acta 163, 188-203 6 Venter, J.C., Schaber, J.S., U'Prichard, D.C. and Fraser, C.M. (1983) Biochem. Biophys. Res. Commun. 116, 1070-1075. 7 Wang, N.I., Fukui, H., Matsuoka, H. and Wada, H. (19861 Biochem. Biophys. Res. Commun. 137, 593-598 8 Kato, I., Kohr, W.J. and Laskowski, M., Jr. (1978) in Regulatory Proteolytic Enzymes and their Inhibitions, l l t h FEBS Meeting (Magnusson, S., Ottesen, M., Foltman, B. Dano, K., Neurath, M., eds.), pp. 197-206, Pergamon
Press, Oxford 9 Kato, I. (1982) Protein, Nucleic Acid Enzyme 27, 1747-1756 10 Watanabe, K., Matsuda, T. and Sato, Y. (1981) Biochim. Biophys. Acta 667, 242-250 11 Matsuda, T., Watanabe, K. and Nakamura, R. (1982) Biochim. Biophys. Acta 707, 121-128 12 Kume, T., Sato, Y. and Umemoto, Y. (1973) Nippon Nogeikagaku Kaishi 47, 549-555 13 Waheed, A. and Salahuddin, A. (1975) Biochem. J. 147, 139-144 14 Shugar, D. (1952) Biochim. Biophys. Acta 8, 302-309 15 Rick, W. and Fritsch, W.P. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.V., ed.), pp. 1046-1053, Academic Press, New York 16 Kato, Y., Komiya, K., Sasaki, H. and Hashimoto, T. (1980) J. Chromatography 190, 297-303 17 Kume, T., Watanabe, H., Takehisa, M. and Sato, T. (1981) Agric. Biol. Chem. 45, 1351-1355 18 Ikenaka, T. (1982) Protein, Nucleic Acid Enzyme 27, 1738-1746 19 Parkinson, T.L. (1966) J. Sci. Food Agric 17, 101-103 20 Lowe, M.E. and Kempner, E.S. (1982) J. Biol. Chem. 257, 12478-12480 21 Beauregard, G. and Potier, M. (1985) Anal. Biochem. 150, 117-120