ARCHIWS OF BIOCHEMISTRY AND BIOPHYSICS Vol. 191, No. 1, November, pp. 119-124,1978
Value Based on the Prosthetic Ascorbate Oxidase
R. DAWSON New York, Neu! York 10027
Received March 3, 1978; revised May 16, 1978 The copper content and activity data of 137 purified samples of ascorbate oxidase (Lascorbate:Os oxidoreductase, EC 22.214.171.124) prepared in this laboratory during the period 1951-1977 have been examined and correlated. These data support the developing concepts of “active site heterogeneity” in otherwise homogeneous protein preparations. The specific activities, based on the copper contents of these 137 enzyme specimens, have been determined to average at about 760 units per pg copper and to reach maximum values in the area of 1000 units per pg copper. The maximum specific activity value (units per mg protein) and the copper content value of these purified specimens have been found to be 3800 + 400 and 0.46 + 0.06’S, respectively.
A previous investigation (1, 2) concerned with the purification and characterization of the copper protein, ascorbate oxidase (L-ascorbate:Oz oxidoreductase, EC 126.96.36.199) focused attention on the fact that over a period of 23 years the specific activity values and the copper content values of homogeneous preparations of the enzyme greatly increased. As can be seen in Table I, the 1973 specific activity values per mg protein approximately double those that were characteristic of the first homogeneous preparation of the enzyme described in 1951 (4). It is particularly pertinent that during this extended period, involving the ,purification of 129 specimens of the enzyme, no significant variation was observed in the molecular weight, the degree of homogeneity, and the amino acid composition of the protein moiety of the purified oxidase (1, 4-6). This fact has provided a basis for the view (1) that the earlier homogeneous preparations (4-16) of lower copper content may have been the result of varying degrees of “prosthetic copper loss” occurring during the purification processes -then employed. Consequently, it has seemed advisable to ’ Present address: Department of Pharmacology, Yale Universit,y School of Medicine, New Haven, Conn. 06510.
examine, from this point of view, the copper and activity data of the purified samples of the enzymes prepared in this laboratory during the past 27 years. Of particular interest was an evaluation of the average catalytic activity value per pg of total prosthetic copper, and the maximum value achievable for the catalytic activity per pg of prosthetic copper. The results of the survey, involving 137 enzyme preparations (eight additional preparations during the period 1973-1977) ranging in copper content from 0.20 to 0.57% are shown in chronological order in Fig. 1. All of the enzyme preparations involved have been judged to be homogeneous, or nearly homogeneous, by the electrophoretic and ultracentrifugal criteria of several previous workers (1, 4, 6). Some preparations having specific activity values lower than 500 units per mg protein have been judged partially inactivated or denatured and, therefore, have not been included in this figure. Inasmuch as the prosthetic copper of ascorbate oxidase is labile to various agents and environmental factors (l), including particularly the conditions and time of freezer storage of the raw materials, a few enzyme specimens prepared by the current procedure (1) have not been included because of low enzyme quality. 19 0003-9861/78/1911-0119$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
A COMPARISON OF HOMOGENEOUS ASCORBATE OXIDASE PREPARATIONS’ Investigators Specific activity Molecular Copper content in units Weight
Dunn and Dawson Lee and Dawson
per w protein
.per PLg ‘copper
146,000 -c 10% 140,000
u One unit of ascorbate oxidase activity has been defined as the amount of enzyme that causes an initial rate of oxygen uptake of 10 jd per min under the described conditions (3).
__-=z rz 5
a 0: ii’ -r g ‘E $2
n YEAR NUMBER PREPARATlONS
‘54 ‘56 6
FIG. 1. A chronological correlation of the copper specific activities (units per ug copper) and the copper content of 137 homogeneous ascorbate oxidase specimens prepared in this laboratory during,the period 1951-1977. The upper part of the figure reveals the average copper contents (solid bars) and their standard deviations (spaced bars). The lower figure shows (by lower broken line) the average copper specific activity of the annual groups of purified enzyme specimens (solid bars). Each dot above the solid bars represents a preparation having a specific activity value higher than 800. These dots reveal the frequency of preparations having copper specific activities approaching maximum. The upper broken line at 1000 represents an approximation of the maximum value.
When the specific activity of the enzyme is examined as a function of the copper content, the results shown in Fig. 2 are obtained. The data in this figure are taken from 167 oxidase preparations which in-
eluded the 137 homogeneous preparations of Fig. 1 and 30 specimens that had been only partially purified by earlier procedures. The data as arranged in Fig. 1 reveal
0.13- 0.180.17 022
FIG. 2. Correlation of the copper contents and protein ascorbate oxidase preparations. The solid bars represent the spaced bars indicate their standard deviations.
that, whereas the copper content of the enzyme preparations increased progressively during the 27-year period from 0.23 + 0.02% to 0.46 f 0.06%, the catalytic activity per pg of the prosthetic copper remained close to an average value of 760 units per pg of copper (lower broken line). The number of preparations having a specific activity greater than 800, as represented by the dots (plus six not shown) in Fig. 1, correspond to 51 preparations (37.2% of the total). The data of Fig. 1 also reveal that the maximum catalytic activity value approaches 1000 units per pg of copper (upper broken line). From Fig. 2, it is apparent that the specific activity (units per mg protein) of the enzyme increases as a function of the copper content of the enzyme. The data justify a conclusion that the maximum specific activity of the enzyme is 3800 units per mg of protein with a deviation of about f 400 units.
specific activities (units per mg) of 167 the average specific activity value and
It has long been established that ascorbate oxidase is a copper-containing globular protein and the specific activity of the enzyme parallels the copper content of such preparations (4). The results of the present survey support the view that the increase in the specific activity of the more recent enzyme preparations is attributable to a higher content of prosthetic copper rather than to a difference in the protein moiety. It should also be noted that there has been a significant variation in the copper specific activity value of the enzyme over the years (see Fig. 1). This variation in the catalytic efficiency of the prosthetic copper of the enzyme may be dependent on factors other than just the copper content. In recent years, a number of investigations have reported that the blue oxidases ascorbate oxidase, lactase, and ceruloplasmin all contain three forms of prosthetic copper (2, 22-28). All members of the blue
oxidases have many similar molecular properties and the stoichiometry and the state of copper in these enzymes have been reviewed (29-32). According to Deinum et al. . (23), purified ascorbate oxidase preparations from squash contain eight gram atoms of copper and the ratio of type 1, type 2, and type 3 copper, as defined by Malkin and Mahnstrom (29), is 3:1:4. However, there is much evidence that the blue oxidases vary in their content and distribution of copper types (26,27,31,33-35). Investigations on lactase have revealed that whereas type 1 copper is responsible for the characteristic intense blue color of the enzyme (25), type 2 copper is indispensable for catalytic activity (36, 37) and is also the binding site for certain anion inhibitors (27, 37). The nonparamagnetic type 3 copper is believed to act as an oxidation-reduction-active two-electron-accepting unit and is associated with an absorption band in the 330 nm region (27, 38). A study with fungal lactase has also revealed that type 2 copper can be reversibly removed from the native enzymes (37). For ceruloplasmin, there is evidence supporting both a 2:l and a 3:l ratio of type 1 to type 2 copper (39) and these ratios cannot be considered independent of the total content of copper per molecule. In addition, the electron paramagnetic resonance data on ceruloplasmin have shown that the content of type 2 copper can vary between one and two ions per molecule (26). Dawson et al. (32) have pointed out that because of the lability of type 2 copper compared with types 1 and 3, the quality of enzymes used in experimental work is of much importance. The difference in ratio of the three types of copper is at least one of the factors contributing to the difference in quality of the enzyme preparations. Thus, it appears that the variation in copper content of the ascorbate oxidase preparations that has been observed in this laboratory (see Fig. 1 and Table I) is associated with a variation in quality of the enzymes. The copper-specific activity of each enzyme preparation is a reflection of the ratio of the type 1 and type 3 to the type 2 copper in each enzyme. A recent investigation of anion binding to ascorbate oxidase in this laboratory (40)
reveals that both azide and fluoride bind to type 2 copper and that this copper is also part of an ascorbte binding site. Also, the lability of catalytically active type 2 copper and its strong affinity for certain anions that results in inactivation and inhibition of the enzyme (40) are possible factors that contribute to the variation in catalytic efficiency of the copper in purified ascorbate oxidase preparations. The catalytic mechanism of a blue oxidase is thus complex. It has been proposed, particularly for Polyporus lactase, that all three types of copper co-operate in the catalytic function of the enzyme (36, 38). The catalytic mechanism by means of which the three enzymatic copper types are involved in an intramolecular electron transfer with the substrate has been reviewed for the three blue oxidases by Gray and co-workers (41). There has been increasing evidence in recent investigations to support the view that “protein homogeneity” does not necessarily guarantee “active site homogeneity.” The most convincing examples of this evidence have been derived from studies of metal enzymes where the active site has a distinctive marker. The data given in the present report support the conclusion reached by other investigators (42) that a finite stoichiometry of the various copper types in the larger copper proteins cannot be defined at this time. Furthermore, the data of this report support the developing concepts of “active site heterogeneity” in otherwise apparently homogeneous protein preparations. In addition to the blue copper oxidases, other examples of active site heterogeneity are found in other metalloenzymes such as succinate dehydrogenase and xanthine oxidase. Thus, in similarity to ascorbate oxidase, a variation in the metal content of succinate dehydrogenase, accompanying a change in specific activity value and no obvious difference in regard to electrophoretic or ultracentrifugal behavior, has been observed (43-45). Solubilized succinate dehydrogenases from mammalian tissues (46-49) and from Rhodopseudomonas sphaeroides (50) have been found to contain variable proportions of three iron-sulfur centers. The presence of an additional iron-sulfur center of the fer-
redoxin type in Rhodospirillum rubrum succinate dehydrogenase has been very recently reported (51). Similarly, the iron of milk xanthine oxidase is heterogenic. The enzyme gives two types of electron paramagnetic resonance signals attributed to iron-sulfur systems (52). REFERENCES 1. LEE, M. H., AND DAWSON, C. R. (1973) J. Biol. Chem. 248, 6596-6602. 2. LEE, M. H., AND DAWSON, C. R. (1973) J. Biol. Chem. 248, 6603-6609. 3. DAWSON, C.R., AND MAGEE, R. J. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, pp. 831-835, Academic Press, New York. 4. DUNN, F. J., AND DAWSON, C. R. (1951) J. Biol. Chem. 189, 485-497. 5. STARK, G. R., AND DAWSON, C. R. (1962) J. Biol. Chem. 237, 712-716. 6. TOKUYAMA, K., CLARK, E. E., AND DAWSON, C. R. (1965) Biochemistry 4, 1362-1370. 7. JOSELOW, M., AND DAWSON, C. R. (1951) J. Biol. Chem. 191, l-10. 8. MAGEE, R. J., AND DAWSON, C. R. (1954) Arch. Biochem. Biophys. 99, 338-347. 9. KIRSCHENBAUM, D. M. (1956) Ph.D. dissertation, Columbia University, New York, N.Y. 10. GREENWALD, B. (1962) Ph.D. dissertation, Columbia University, New York, N.Y. 11. POILLON, W. N., AND DAWSON, C. R. (1963) Biochim. Biophys. Acta 77, 27-36. 12. PENTON, Z. G. (1964) Ph.D. dissertation, Columbia University, New York, N.Y. 13. TANG, S. P. W. (1966) Ph.D. dissertation, Columbia University, New York, N.Y. 14. WILSON, J. B. (1966) Ph.D. dissertation, Columbia University, New York, N.Y. 15. CLARK, E. E., POILLON, W. N., AND DAWSON, C. R. (1966) Biochim. Biophys. Acta 118, 72-81. 16. CLARK, E. E., POILLON, W. N., AND DAWSON, C. R. (1966) Biochim. Biophys. Acta 118, 82-87. 17. LEE, M. H. (1968) Research note, Columbia University, New York, N.Y. 18. LEE, M. H. (1968) Ph.D. dissertation, Columbia University, New York, N.Y. 19. CHANG, H. T. (1969) Ph.D. dissertation, Columbia University, New York, N.Y. 20. STROTHKAMP, K. G. (1973) Ph.D. dissertation, Columbia University, New York, N.Y. 21. KRUL, K. G. (1973) Ph.D. dissertation, Columbia University, New York, N.Y. 22. AVIGLIAKO, L., GEROSA, P., ROTILIO, G., FINAZZI AGRO, A., CALABRESE, L., AND MONDOVI, B. (1972) Ital. J. Biochem. 21, 248-255. 23. DEINUM, J., REINHAMMAR, B., AND MARCHESINI,
A. (1974) FEBS Lett. 42, 241-245. 24. BROMAN, L., MALMSTRBM, B. G., AASA, R., AND VANNGARD, T. (1962) J. Mol. Biol. 5, 301-310. 25. MALMSTR~M, B. G., REINHAMMAR, B., AND V.&NNGARD, T. (1968) Biochim. Biophys. Acta 156,67-76. 26. DEINUM, J., AND VKNNGARD, T. (1973) Biochim. Biophys. Acta 310, 321-330. 27. MALMSTR~~M, B. G., REINHAMMAR, B., AND V~~NNGARD, T. (1970) Biochim. Biopkys. Acta 205.48-57. 28. MAKINO, N., AND OGURA, Y. (1971) J. Biochem. (Tokyo) 69,91-100. 29. MALKIN, R., AND MALMSTR~M, B. G. (1970) Adu. Enzymol. 33, 177-243. 30. VANNGARD, T. (1972) in Biological Applications of Electron Spin Resonance (Swartz, H. M., Bolton, J. R., and Borg, D. C., eds.), pp. 411-447, John Wiley & Sons, N. Y. 31. MALMSTROM, B. G., ANDR~ASSON, L.-E., AND REINHAMMAR, B. (1975) in The Enzymes, Ed. 3, Vol. 12, pp. 507-579. 32. DAWSON, C. R., STROTHKAMP, K. G., AND KRUL, K. G. (1975) Ann. N. Y. Acad. Sci. 258.209-220. 33. OMURA, T. (1961) J. Biochem. (Tokyo) 50, 264-272. 34. REINHAMMAR, B. (1970) Biochim. Biophys. Acta 205, 35-47. 35. MCKEE, D. J., AND FRIEDEN, E. (1971) Biochemistry 10,3880-3883. 36. MALKIN, R., MALMSTROM, B. G., AND VANNGARD, T. (1968) FEBS Lett. 1, 50-54. 37. MALKIN, R., MALMSTR~M, B. G., AND VANNGARD, T. (1969) Eur. J. Biochem. 7,253-259. 38. MALKIN, R., MALMSTR~M, B. G., AND V;~NNGARD, T. (1969) Eur. J. Biochem. 10.324-329. 39. WEVER, R., VAN LEEUWEN, F. X. R., AND VAN GELDER, B. F. (1973) Biochim. Biophys. Actu 302, 236-239. 40. STROTHKAMP, R. E., AND DAWSON, C. R. (1977) Biochemistry 16, 1926-1929. 41. HOLWERDA, R. A., WHERLAND, S., AND GRAY, H. B. (1976) Annu. Rev. Biophys. Bioeng. 5, 363-396. 42. BEINERT, H. (1977) Coor. Chem. Reu. 23.119-129. 43. GREEN, D. E., MII, S., AND KOHOUT, P. M. (1955) J. Biol. Chem. 217, 551-567. 44. SINGER, T. P., KEARNEY, E. B., AND BERNATH, P. (1956) J. Biol. Chem. 223, 599-613. 45. SINGER, T. P., KEARNEY, E. B., AND MASSEY, V. (1956) in Internationl Symposium. Enzymes: Units of Biological Structure and Function (Gaebler, 0. H., ed.), pp. 417-432, Academic Press, New York. 46. BEINERT, H., ACKRELL, B. A. C., KEARNEY, E. B., AND SINGER, T. P. (1975) Eur. J. Biochem. 54, 185-194. 47. OHNISHI, T., SALERNO, J. C., WINTER, D. B., LIM, J., Yu, C. A., Yu, L., AND KING, T. E. (1976) J.
Biol. Chem. 251,2094-2104. 48. OHNISHI, T., LIM, J., WINTER, D. B., AND KING, T. E. (1976) J. Biol. Chem. 251,2105-2109. 49. SALERNO, J. C., OHNISHI, T., LIM, J., AND KING, T. E. (1976) Biochem. Biophys. Res. Commun. 73,833~840.
DAWSON 50. INGLEDEW, W. J., AND PRINCE, R. C. (1977) Arch. Biochem. Biophys. 178,303-307. 51. CARITHERS, R. P., YOCH, D. C., AND ARNON, D. I. (1977) J. Biol. Chem. 252, 7461-7467. 52. LOWE, D. J., LYNDEN-BELL, R. M., AND BRAY, R. C. (1972) Biochem. J. 130.239-249.