Acceleration of the Rate of Deamidation of GlyArgAsnArgGly and of ...

Proc. Nat. Acad. Sci. USA Vol. 70, No. 7, pp. 2122-2123, July 1973

Acceleration of the Rate of Deamidation of GlyArgAsnArgGly and of Human Transferrin by Addition of I-Ascorbic Acid (molecular "timer"/nutrition/vitamin

ARTHUR B. ROBINSON, KAREN IRVING, AND MARY McCREA Department of Biology, University of California, San Diego, La Jolla, Calif. 92037

Communicated by Martin D. Kamen, March 16, 1973 ABSTRACT Experiments on the model peptide, GlyArgAsnArgGly, and the protein, human transferrin, have shown that hydrolytic deamidation of these molecules is markedly accelerated by addition of physiologically significant concentrations of 1-ascorbic acid. Since hydrolytic deamidation has been suggested as an important timer of biological events, the effects on hydrolytic deamidation of substances that are normally present in living organisms and are subject to nutritional control are of special relevance.

The general biological importance of hydrolytic deamidation molecular "timer" in living things has been suggested by Robinson, MlcKerrow, and Cary (1). Several specific biological roles for hydrolytic deamidation have been suggested (2-4). Flatmark (5-8) has characterized the naturally occurring deamidated forms of horse-heart and rat cytochrome c, and numerous examples of hydrolytic deamidation of proteins have been studied. If, as has been suggested (1), hydrolytic deamidation of glutaminyl and asparaginyl residues is a generally important molecular timer of biological processes, then variations of deamidation rate with variations in concentration of the substances normally present in living organisms is of great importance. Of special interest are those substances that affect deamidation rate and are subject to dietary control (9-11). We have begun a survey of the effects on deamidation rate of some of the small molecules that are required for life. We have found that addition of I-ascorbic acid markedly increases the hydrolytic deamidation rates of the peptide, GlyArgAsnArgGlv, and the protein, human transferrin. This is a qualitatively new observation, and these experiments are summarized in Figs. 1 and 2. I-Ascorbic acid is distributed in various concentrations in most living tissues. Concentrations of i-ascorbic acid have been reported up to 4.3 mM in human tissues (12-14), up to 8.5 mM in guinea pig tissues (15-17), up to 4.3 mMI in rat tissues (18), up to 0.1 mMA in hen plasma (19), and up to 11.6 mM in plants (14, 20). We have demonstrated that addition of i-ascorbic acid at these concentrations increases the rate of hydrolytic deamidation. These concentrations of i-ascorbic acid could, therefore, change biological processes in which deamidation is important. The properties of in vitro solutions are, of course, somewhat different from those of in vivo solutions. Deamidation of simple peptides is first-order in l)eptide concentration (1). However, when i-ascorbic acid is present the first-order rate of deamidation of Gly*ArgAsnArgGly decreases with time. The rate of deamidation of human trans2122 as a

our

ferrin also decreases with time (Fig. 2). This decrease is slower for higher initial concentrations of i-ascorbic acid. Transferrin deamidation is complicated because several amides are deamidated and because changes in the tertiary structure of transferrin are probably involved. We analyzed human transferrin that had been deamidated by 0.01 formal (F), I-ascorbic acid solutions by polyacrylamide gel electrophoresis (21). This analysis indicated that the product increases in heterogeneity and in negative charge with time. More than 99% of the transferrin molecules were partially deamidated after 3 days. Since the concentration of i-ascorbic acid decreases with time in these aerobic solutions, it may be that an oxidation product of i-ascorbic acid rather than i-ascorbic acid itself is responsible for the increased rate of deamidation observed in the experiments described above. Our purpose here is to report the dependence of deamidation i-ascorbic acid in aerobic solutions of biological interest.

on

MATERIALS AND METHODS

Gly*ArgAsnArgGly was synthesized by Merrifield solid-phase

peptide synthesis (22-27). The peptide

was

purified

on

Sephadex G-10 in pyridine acetate buffer (pH 7) and freezedried. 0.8-mM Solutions of GlyArgAsnArgGly in sodium phosphate buffer of ionic strength 0.15 (pH 7.0) were sealed in glass tubes after addition of various amounts of i-ascorbic acid (Hoffman-LaRoche crystalline). The tubes were main-

0

vX

20 7-

0

0.03 0.01 0.02 Fornality of /-ascorbic acid

0.04

FIG. 1. Experimental values for the time required for 50% deamidation of the peptide Gly*ArgAsnArgGly in aerobic solutions of different formalities of ascorbic acid. All values are for 0.8 mMI solutions of peptide in sodium phosphate buffer (pH 7.0), 0.15 ionic strength, at 37°. The value at 0.8 mF ascorbic acid was obtained by extrapolation of a series of measurements up to 30% deamidation. All other values were obtained by exponential interpolation of measurements above and below 50% deamidation.

Proc. Nat. Acad. Sci. USA 70

(1973)

Deamidation of GlyArgAsnArgGly and of Human Transferrin

2123

DISCUSSION

Aerobic solutions of i-ascorbic acid markedly increase the rate of deamidation of Gly*ArgAsnArgGly and of human transferrin. It may be that an oxidation product of i-ascorbic acid is responsible for this rate increase. The increased rate of deamidation of Gly*ArgAsnArgGly and human transferrin in the presence of physiologically significant concentrations of 1-ascorbic acid is interesting because deamidation may play a central role as a timer in develop-

Days

FIG. 2. Measurements of NH3 evolved from a 27 ,uM solution of human transferrin were made with no i-ascorbic acid, V -V; and 0.1 mF, -O; 0.5 mF, O EO; 1 mF A A; and 3 mF solutions of l-ascorbic acid, XX. All values are for sodium phosphate solutions (pH 7.42), 0.2 ionic strength, at 370, containing 16 g of kanamycin sulfate per ml and 16 g of neomycin sulfate per ml.

tained at 37°. The amino-terminal glycyl residue was labeled in its carboxyl carbon with 14C. Periodically, a tube was opened and its contents were applied to a paper electrophoresis strip. Electrophoresis was performed for 3 hr at 400 V in 0.01 M (pH 5.0) pyridine acetate buffer. The resulting bands were eluted with 1% ammonia solution and assayed in a liquid scintillation counter. Human transferrin was obtained from the Research Products Division of Miles Laboratories Incorp. and was used without further purification. Ammonia concentrations were measured by a modified (28) ion-exchange technique (29). All glassware and pipettes used in the transferrin experiments were washed carefully, rinsed in glass-distilled water and ammonia-free water, and stored in an oven at 90(100'. They were removed from the oven just before use. Ammonia-free water, made by passing glass-distilled water through a column of Durrum DC-3 resin obtained from Durrum Chemical Corp., Palo Alto, Calif., was used for all solutions in the transferrin experiments. Fresh reagents for ammonia determinations were made daily. Absorbances were measured by means of a Zeiss PMQII spectrophotometer. A sodium phosphate buffer (pH 7.42), ionic strength 0.2, containing 16 mg of kanamycin sulfate per ml and 16 mg of neomycin sulfate per ml was used for all of the transferrin experiments. Each day a 27-MM solution of human transferrin (3Q,*) was made in the buffer solution of interest. A 4-ml aliquot of this solution was put into the lower reservoir of a Thunberg tube, to the upper reservoir of which five drops of ion-exchange resin (28) had previously been added. All tubes were prepared in duplicate and incubated at 37°. After incubation, the tubes were brought to room temperature (220) and the resin was washed into the lower reservoir. Solutions were prepared at such times that all of the tubes for a given experiment were removed from incubation at 370 and measured for ammonia (28) on the same day. We used 2 mg of human transferrin per ml and assumed that human transferrin has a molecular weight of 74,000. *

ment, in protein turnover, and in aging (1). Differences in the state of health associated with differing dietary intakes of i-ascorbic acid (10, 11) could be rationalized as owing to changes in some of the timed processes that are essential to biological function. We thank Prof. M. D. Kamen and L. Pauling for helpful discussions and encouragement. This work was supported in part by USPHS Grant AM 14879 to A.B.R. and Grant GM 18528 to M. D. Kamen. 1. Robinson, A. B., McKerrow, J. H. & Cary, P. (1970) Proc. Nat. Acad. Sci. USA 66, 753-757. 2. Palmer, W. G. & Papaconstantinou, J. (1969) Proc. Nat. Acad. Sci. USA 64, 404-410. 3. Sinex, F. M. (1960) J. Gerontol. 15, 15-18. 4. Wherrett, J. R. & Tower, D. B. (1971) J. Neurochem. 18, 1027-1042. 5. Flatmark, T. (1964) Acta Chem. Scand. 18, 1517 and 1656. 6. Flatmark, T. (1966) Acta Chem. Scand. 20, 1487-1496. 7. Flatmark, T. (1967) J. Biol. Chem. 242, 2454-2459. 8. Flatmark, T. & Sletten, K. (1968) J. Biol. Chem. 243, 16231629. 9. Pauling, L. (1968) Science 160, 265-271. 10. Pauling, L. (1971) Vitamin C and the Common Cold (W. H. Freeman and Co., San Francisco). 11. Stone, I. (1972) The Healing Factor (Grosset and Dunlap). 12. Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 13. Melka, J. (1936) Arch. Gesamte Physiol. Menschen Tiere 237, 216-221. 14. Wagner, Von F., Hofmann, K. D., Preibosh, W. & Koob, G. (1970) Zentralbl. Gynaekol. 92, 1060-1061. 15. Schaffert, R. R. & Kingsley, G. R. (1955) J. Biol. Chem. 212, 59-68. 16. Zloch, Z. & Ginter, E. (1970) Z. Klin. Chem. Klin. Biochem. 8, 302-305. 17. Penney, J. R. & Zilva, S. S. (1946) Biochem. J. 40, 695-706. 18. Hughes, R. E., Hurley, R. J. & Jones, P. R. (1971) Exp. Eye Res. 12, 39-43. 19. Allison, J. H. & Stewart, M. A. (1971) Anal. Biochem. 43, 401-409. 20. Dorr, P. E. & Nockels, C. F. (1971) Poultry Sci. 50, 13751382. 21. Barakat, M. Z., Shehab, S. K., Daruish, N. & Afify, N. (1970) Bull. Acad. Pol. Biol. 18, 1-5. 22. Merrifield, R. B. (1964) J. Amer. Chem. Soc. 86, 304-305. 23. Marshall, G. R. & Merrifield, R. B. (1965) Biochemistry 4, 2394-2401. 24. Marglin, A. & Merrifield, R. B. (1966) J. Amer. Chem. Soc. 88, 5051-5052. 25. Stewart, J. M. & Young, J. D. (1969) Solid Phase Pe'ptide Synthesis (W. H. Freeman and Co., San Francisco). 26. Sakakibara, S., Shimonishi, Y., O'Kada, M. & Koshida, Y. (1966) Peptides Proc. Eur. Symp. 8th. 27. Leonard, J. & Robinson, A. B., J. Amer. Chem. Soc. 89, 181-182. 28. Hyland Division of Travenol Laboratories Incorporated, Costa Mesa, California, Technical Bulletin D-60/32-5214G, revised September (1968) 29. Forman, D. T. (1964) Clin. Chem. 10, 497-508. 30. Roberts, R. C., MaKey, D. G. & Seal, U.S. (1966) J. Biol. Chem. 241, 4907-4913.