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OCR for page 92
92 PART II. BIOSYNTHESIS OF HEMOGLOBIN 11. Bogorad, L.: Intermediates in the biosynthesis of porphyrins from porphobilinogen, Science 121: 878, 1955. 12. Rimington, C.: Haems and porphyrins in health and disease, I., Acta Med. Scand. 143: 161, 1952. 13. Mauzerall, D., and Granick, S.: Uroporphyrinogen decarboxylase. In preparation. 14. Watson, C. G., deMillo, R. P., Schwartz, S., Hawkinson, V. E., and Bossenmaier, T.: Porphyrin chromogens or precursors in urine, blood, bile, and feces, J. Lab. and Clin. Med. 37: 831, 1951. DISCUSSION Dr. Herbert C. Schwartz: We have heard Dr. Shemin's, Dr. Bogorad's and Dr. Granick's interesting accounts of their studies on the enzymes in- volved in biosynthesis of heme. I would like to tell you briefly about some of our studies. Dr. Cartwright, Dr. Wintrobe and I have studied the biosynthesis of heme from protoporphyrin and iron in a chicken hemolysate system. The per cent of added radioiron incorporated into heme (per cent uptake of Fe59) was used as a measure of heme synthesis. Our studies have suggested that this reaction is enzyme dependent. In a typical experiment, the incubation of hemolysate + protoporphyrin + Fe59 gave 13.5 per cent uptake of Flew, whereas pro- toporphyrin + Fe59 (without hemolysate) or hemolysate + Few (without protoporphyrin) gave less than 1 per cent uptake of :Fe59. Prom such a hemolysate an active, particle-free preparation was obtained by making the hemolysate isotonic with potassium chloride and homogenizing for 10 minutes in a Waring Blendor, as suggested by Dr. Shemin. The super- nate, after centrifugation at 100,000 x G. had two to three times the activity of the original hemolysate. Subsequent studies were performed on this soluble preparation. 50 4 30 A: 20 10 o 1 1 1 7.5 8.0 8.5 9.0 pH FIG. 1. Relation of uptake of Fe59 to pH. ?~ j i: \ ~ 10W / s _ Let/ 1 1 1 1 1 \t . 1. 4 5 6 7 8 9 10 P ~ FIG. Z. --Stability of iron uptake related to pH over 3-hour period.

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DISCUSSION ~3 Optimal activity (fig. 1) was at pH 7.8. Optimal stability for three hours (fig. 2) was at pH 7.4. Time studies (fig. 3) showed the per cent uptake of Fe59 to be maximal in three to four hours and to be a linear function of time over the first 30 minutes. The rate of heme synthesis at 30 minutes was pro- portional to enzyme concentration. The preparation was not storable at 5 C., - 3(~ C., or lyophilized and was found to be inactivated after heating at 56 C. for 30 minutes. 35: 30 25 2C _ A ;~1^ _ 0 1 1 1 2 3 TIME tnourl) 1 / 1 1 4 S FIG. 3. Uptake of Fe5'0 with time. The effect of thiol inhibitors is shown in table I. There was marked in- hibition with 1 x 10-M p-chloromercuribenzoate (PC~:B) and 1 ~ 10-2M iodoacetamide, and slight inhibition with ~ x 10-3M p-chloromercuribenzoate and ~ x 10-3M iodoacetamide. TABLE I EFFECT OF THIOL INHIBITORS do Uptake Fe59 RIO Inhibition Control 3 S PCME ( 1 x 10 - 2M) 7 80 PCME (1 x 10 - 3M) 33 7 PCMB (1 x 10 - 4M) 35 0 lodoacetamide ( 1 ~ 10 - 2M) 22 37 Todoacetamide ( 1 x 10 - 3M) 32 9 fodoacetamide ( 1 ~ 10 - 4M) 3 S 0 When dialyzed against water for 22 hours (table II), there was a 70 per cent loss of activity. The original activity could be restored by reconsti- tution of the dialyzed sample with a heat-inactivated preparation or by the addition of 1 x 10-~M glutathione. While these studies were in progress, Krueger, ~:elnick and Klein inde- pendently published similar studies on the biosynthesis of heme from iron and protoporphyrin. Except for the dialysis data, their findings are essentially in agreement with ours,

OCR for page 92
94 PART II. BIOSYNTHESIS OF HEMOGLOBIN TABLE II % Uptake Few Experiment A Experiment B Control 29 52 Dialyzed (INTO) 9 16 Reconstituted 27 Glutathione ( 1 ~ 10 - 3M) 19 Glutathione ( 1 x 10~M) 52 REFERENCES 1~ Goldberg, A., Ashenbrucker, H., Cartwright, G. E., and Wintrobe, M. M.: Studies on the biosynthesis of heme in vitro by Adrian erythrocytes, Blood 11: 821, 1957. 2. Schwartz, H. C., Cartwright, G. E., and Wintrobe, M. M.: Studies on the syn- thesis of heme from protoporphyrin, Clin. Research Proceedings 5: 29, 1957. 3. Shemin, D., Abramsky, T., and Russell, C. S.: The synthesis of protoporphyrin from delta-amino levulinic acid in a cell-free extract, J. of the Am. Chem. Soc. 76: 1, 1954. 4. Krueger, R. C., Melnick, I., and Klein, J. R.: Formation of heme by broken-cell preparations of duck erythrocytes, Arch. of Biochem. and Biophys. 64: 302, 1956.