Ayers, Valborg E.

Hoffman, Peg

Morell, Samuel A.

Taketa, Fumito

English

PubMed

epublications@Marquette

Marquette Universitye-Publications@MarquetteBiomedical Sciences Faculty Research andPublications Biomedical Sciences, Department of6-1-1962Reversible Changes in the NEM-reactive--SHGroups of Hemoglobin on Oxygenation-deoxygenationSamuel A. MorellMarquette UniversityPeg HoffmanMilwaukee Blood CenterValborg E. AyersMilwaukee Blood CenterFumito TaketaMilwaukee Blood CenterPublished version. Proceedings of the National Academy of Sciences of the United States of America, Vol.48, No. 6 ( June 1962): 1057-1061. Permalink. © 1962 National Academy of Sciences. Used withpermission.REVERSIBLE CHANGES IN THE NEM*-REACTIVE -SH GROUPSOF HEMOGLOBIN ON OXYGENATION-DEOXYGENATIONtBY SAMUEL A. MORELL, PEG HOFFMAN, VALBORG E. AYERS, AND FUMITOTAKETADEPARTMENTS OF BIOCHEMISTRY, MILWAUKEE BLOOD CENTER, INC., AND MARQUETTE UNIVERSITYSCHOOL OF MEDICINE, MILWAUKEE, WISCONSINCommunicated by Linus Pauling, April 17, 1962In a previous communication the reaction of N-ethylmaleimide (NEM) with thethiols of intact human erythrocytes was described.1 The principal sites of reactionare the p-chains of hemoglobin2 and intracellular glutathione. Average values of2.35 and 0.45 equivalents of HbSH and GSH respectively per mole of hemoglobinhave been found for normal red cells. The conditions used were NEM/Hb = 8,-1 hr, 250C, pH 6.8. NEM is relatively unstable above pH 7 and reacts readilywith thiols at pH 6.8.3 As shown in Figure 1, the uptake of NEM by both erythro-3.0e- ~~~~GSH2.5 _2.0J0Uj 1.0 ,0.5 ,10 20 30 40 50 60 70M N U T E SFIG. 1.-Rate of NEM-reaction with intact erythrocytes and dialyzed stroma-freehemolysates. NEM 0.002 M, pH 6.8, 23 1 10C, molar ratio NEM/Hb = 8. See Table1 for procedure and calculation of equiv-SH/mole Hb, uncorrected for GSH.cytes and dialyzed hemolysates, measured by decrease in absorption at 300 m/,is rapid during the first 30 minutes and very slow after one hour. The reagent ex-hibits a relatively high degree of specificity in reacting rapidly with -SH groups.3-8No significant amounts of NEM are bound when the -SH groups of hemoglobinare alkylated8 and the measurement of -SH by amperometric titration has beencorrelated with OD3oo.2 Reactions with other sites on the protein, as well as theimidazole-catalyzed polymerization of NEM,9 are apparently secondary. Correla-tions of OD)oo with S-cysteinosuccinic acid formed on hydrolysis of NEM-treatedproteins also indicate that the rapid reaction of NEM with proteins is limitedto condensation with -SH groups.7' 10The purpose of the present communication is to report evidence that the reactive- SH groups of hemoglobin undergo reversible changes on oxygenation-deoxygena-,tion. As measured by reaction of NEM with erythrocytes for one hr at pH 6.8,the number of -SH groups decreased on deoxygenation from about 2 (2.3 ± 0.2) to10571058 BIOCHEMISTRY: MORELL ET AL. PROC. N. A. S.TABLE 1DECREASE IN THE NEM-REACTIVE -SH GROUPS OF HEMOGLOBIN ON DEOXYGENATINGERYTHROCYTESa---Oxygenated---- -- Deoxygenated------Blank Ao Bo Co Am BoCoVml buffer, pH 6.811 5 3 4 4 3 4 4ml RBC suspn 0 1 0 1 1 0 130 min, 230 C gas with N2ml NEM, 0.01 M, pH 6.8 0 1 1 0 1 1 0OD300 0.423 0.601 0.041 0.539 0.601 0.045AOD3oo 0.219 0.107equiv (HbSH + GSH)/mole Hb 2.71 1.32equiv -SH/mole Hb 2.23 0.84A washed suspension of human erythrocytes (hematocrit, 25.0%; Hb, 8.6%; GSH, 19.2 mg %; moles GSH/mole Hb, 0.48) was reacted with an excess of NEM before (Ao tube) and after (Aso tube) deoxygenation with ni-trogen for 30 min. Subscripts refer to minutes of bubbling nitrogen through the solutions (1 mm I.D. capil-lary, 200 4 25 bubbles/min). The NEM reaction was conducted in stoppered tubes and all transfers weremade under nitrogen. After 1 hr at 251C, pH 6.8, the tubes were immersed in ice and centrifuged at 4°C (stop-pered). Aliquots of the clear supernates (negligible hemolysis) were mixed with equal volumes of cold 10%perchloric acid, after which precautions regarding reoxygenation were no longer necessary. After centrifuging,the supernates were filtered through Whatman $44 and read in 1 cm cells at 300 mp against the blank. Thebuffer blanks, the NEM tubes (B) and the RBC blanks (C tubes) were treated in an identical manner to ob-tain OD=oo = OD701 tubes B - (A - C). The molar absorbency of NEM at 300 my, pH 6.8, is 6204 and anassumed MW of 66,000 for hemoglobins was used to calculate: equiv - SH/mole Hb = (ODsoo) (66000)/(620)(%Hb) - GSH/rnole Hb.1 (0.9 ± 0.2) equivalents per mole of hemoglobin. The decrease was fully reversibleon reoxygenation. On varying the time of reaction with NEM from 15 min to 24hr, or the NEM/Hb ratio from 3 to 30, the decrease in -SH on deoxygenation re-mained practically constant, 1.2 i 0.2. This decrease, referred to as "the A-SHreaction," was also demonstrated in dialyzed hemolysates. The pH optimum forthe reaction was found to be 6.8-7.0, A-SH being reduced about 30 per cent at pH6.1 and at 7.3.The decrease in reactive -SH groups of hemoglobin on deoxygenating humanerythrocytes is shown in Table 1. On bubbling nitrogen through a washed cellsuspension for 30 min at 250C and pH 6.8, the reactive -SH groups decreased from2.23 to 0.84 equivalents per mole of hemoglobin, 62.3 per cent. A similar ex-periment was conducted on a dialyzed hemolysate, -SH/mole Hb decreasing from2.33 to 1.13, 51.9 per cent.The effect of oxygen saturation on2.4 the magnitude of A-SH and the revers-Z2.2 ibility of the reaction are shown ino0 Table 2. The data are plotted ino Figures 2 and 3 where it may be seenI.l8 that (a) the rate of change of -SH iso .6 greatest during the first 10 min of de-91.4 \ oxygenation, corresponding to the ap-> . proximate range of normal respiration,o '.2 65-95 per cent saturation;'4 (b) in thisl.0 range of oxygenation where approxi-mately 1 mole 02/mole Hb is exchanged0.8 5 0 20 25 30 3 4060 reversibly, A-SH amounts to 0.7 equiv/MINUTES mole Hb, whereas for the entire rangeFIG. 2.-Decrease in the NEM-reactive -SH of oxygenation, involving 4 moles ofgroups of hemoglobin on deoxygenating ery- 02, A-SH increases to 1.4; and (c) thethrocytes by gassing with nitrogen for 5-60 mmn.Data plotted from columns 1 and 3 of Table 2. sigmoid shape of the curve relating theVOL. 48, 1962 BIOCHEMISTRY: MORELL ET AL. 1059TABLE 2REVERSIBLE CHANGES IN THE NEM-REACTIVE -SH GROUPS OF HEMOGLOBIN ON OXYGENATION-DEOXYGENATION OF ERYTHROCYTESRecoveryN! -SH + A-SHthru 02 - SH per A-SH A-SH Reoxygn ASHRBC Saturation* Mole Hb Deoxygn Reoxygn 2.31 0 % Satn(min) (%) (equiv) (equiv) (equiv) (Ye) (equiv)0 100 2.31 1.40t5 85 2.14 0.17 0.10 97.0 1.2310 70 1.68 0.63 0.81 107.8 0.7715 55 1.38 0.93 0.82 95.2 0.4720 40 1.19 1.12 1.06 97.4 0.2830 10 0.92 1.39 1.22 92.6 0.0140 0 0.94 1.37t 1.39 100.960 0 0.89 1.42t 1.29 94.4* Approximated from OD 650/805 at 0, 5, 30 and 60 min N2.1"tA - SH on compete deoxygenation = 1.40 (average of 1.37 and 1.42 at 40 and 60 min).A series of siliconized 15 ml graduated centrifuge tubes was set up for deoxygenation by bubblingwith nitrogen. The procedure was identical to that described in Table 1, using either NEM ("A" and"B" tubes) or pH 6.8 buffer ("C" tubes). After removing the aliquots for mixing with 10% perchloricacid to obtain A-SH Deoxygn, each tube was immediately stoppered for subsequent reoxygenation("A" tubes) or analysis" for % oxygen saturation ("C" tubes). Prior to reoxygenation, the excess NEMwas removed by first aspirating the remaining supernate, under nitrogen, and then washing the cellstwice with pH 6.8 buffer (tubes stoppered during centrifuging). After adjusting with buffer to 4 ml,air was bubbled rapidly through the samples for four min. Reaction with 1 ml 0.01 M NEM at pH6.8 for one hr was then repeated to obtain A-SH Reoxygn.progressive reversible change in Z 1.4A-SH as a function of the degree °of oxygenation simulates that of 0 1.2the oxygen dissociation curve of I.0-hemoglobin. 0.8Data relating the NEM-reac- E8tive -SH groups of human hemo- >0/globin to the oxygenation proc-ess are presently conflicting. 04Benesch and Benesch8 report that I 02reduced as well as fully oxygen-ated hemoglobin binds the same 10 20 30 40 50 60 70 80 90 00amount of NEM, 2.5 moles in 30 % OXYGENATIONmin at pH 7.3, NEM/Hb = 5. FIG. 3.-Reversible changes in the NEM-reactiveRiggs2 however reports a substan- -SH groups of hemoglobin on oxygenation-deoxygena-tial decrease on deoxygenation, 61 tion of erythrocytes at pH 6.8. Data for A-SH versus% oxygenation plotted from columns 2 and 7 of Table± 16percentin2hratpH6.2to 2.7.6, NEM/Hb = 3. Since 2 hrwere required for reduced hemoglobin to exhibit the same extent of NEM-bindingas oxyhemoglobin exhibited in 15 min, Riggs interpreted this difference as a rateeffect. Only one reaction time however, 2 hr, was reported for reduced hemoglobin.In Figure 4 the effect of time on the reaction of NEM with both oxygenated andreduced hemoglobin is shown. A-SH remained constant, 1.1 ± 0.1 equiv./moleHb, over the 24 hr period studied. Although we have confirmed Riggs' observa-tion that reduced and oxyhemoglobin bind the same amount of NEM in 2 hr and15 min respectively (1.5 ± 0.1), it appears that one of the two NEM reactive -SHgroups is masked on deoxygenation.Evidence for a functional relationship between the sulfhydryl groups of human1060 BIOCHEMISTRY: MORELL ET AL. PROC. N. A. S.3 X hemoglobin and the oxygena-tion process has been reportedOXYGEN-a-t -- by Riggs.2 He proposed that*,) the Bohr effect is made up of0 /'t7contributions from at least two>i it syGE tqp;TED "- different groups and suggestedDE. ~ that on oxygenation a hydro-I-SH gen bond is broken between aIl-chain -SH group and anoxygenation-linked acid group.In accordance with this hy-2 3\' 24 pothesis, as shown in Figure 5,HOURS 24 A-SH is reduced to zero in 5FIG. 4.-Rate of NEM-reaction with oxygenated and de- molar urea. The f-chain cys-oxygenated erythrocytes. Deoxygenation by gassing with teines which contain the reac-nitrogen for 40 min. NEM 0.002 M, pH 6.8, 23 4 1C, tive -SH groups are adjacentmolar ratio NEM/Hb = 8 i 1. See Trable 1 for procedureand calculation of equiv -SH/mole Hb. to histidine residues" and H-bonding of these thiol-imida-3r zole groups may be related to= the A-SH reaction. We haveOXYGENATED found however that the effect0 2 - of pH on A-SH differs fromthat reported for the Bohr ef-a _ - fect. The pH optimum forA-H+ is 7.3,8 16 whereas thatl) >O) for A-SH is 6.8-7.0; and atpH 6. 1, where A-H + is reduced___-to zero,'6 A-SH is reduced by2 3 4 5UREA MOLARITY only about 30 per cent. TheFIG. 5.-Effect of urea on the NEM-reactive -SH objective of our present inves-groups of oxygenated and deoxygenated hemoglobin. tigations is to determineDeoxygenation by gassing with nitrogen for 50 min. whether a functional or a coin-I ml of dialyzed stroma-free hemolysate (1.1 i 0.2 X-10-3 M Hb) in "A" and "C" tubes; NEM 0.002 M, pH cidental relationship exists be-6.8, 23 i 10C, molar ratio NEM/Hb = 9 4 2. See tween the ASH reaction ofTable 1 for procedure and calculation of equiv-SH/ intac throcytean tamole Hb. itact erythrocytes and thatportion of the Bohr effect, the"isohydric shift," which participates in normal respiration.We are indebted to S. Raymond Gambino and R. Caseo, St. Luke's Hospital, Milwaukee, Wis-consin, for their cooperation in the measurement of oxygen saturation; to T. J. Greenwalt forhis interest and helpful suggestions; and to M. Moorman and M. Jaeckle for their technicalassistance.* N-ethylmaleimide.t This investigation was supported by a research grant from the National Heart Institute,United States Public Health Service (Grant No. H-4965).1 Morell, S. A., V. E. Ayers, and T. J. Greenwalt, Federation Proc., 18, 290 (1959).2 Riggs, A., J. Biol. Chem., 236, 1948 (1961).3Gregory, J. D., J. Am. Chem. Soc., 77, 3922 (1955).VOL. 48, 1962 BIOCHEMISTRY: TISSIl8RES AND WATSON 10614Alexander, N. M., Anal. Chem., 30, 1292 (1958).5 Roberts, E., and G. Rouser, Anal. Chem., 30, 1291 (1958).6 Benesch, R., R. E. Benesch, M. Gutcho and L. Laufer, Science, 123, 981 (1956).7Leslie, J., D. L. Williams, and G. Gorin, Anal. Biochem., 3, 257 (1962).8 Benesch, R. and R. E. Benesch, J. Biol. Chem., 236, 405 (1961).9 Smyth, D. G., A. Nagamatsu, and J. S. Fruton, J. Am. Chem. Soc., 82, 4600 (1960).10 Riehm, J. P., and J. C. Speck, Am. Chem. Soc., Div. Biol. Chem., Abstracts, p. 34 C, Chicago,Sept. 3-8 (1961).Hendry, E. B., Clin. Chem., 7, 156 (1961).12 Drabkin, D. L., Federation Proc., 16, 740 (1957).13 Deibler, G. E., M. S. Holmes, P. L. Campbell, and J. Gans, J. Appl. Physiol., 14, 133 (1959).14 White, A., P. Handler, E. L. Smith, and D. Stetten, Principles of Biochemistry (2d ed.;New York: McGraw-Hill Book Co. Inc., 1959), p. 666.15 Goldstein, J., G. Guidottig, W. Konigsberg, and R. J. Hill, J. Biol. Chem., 236, PC 77 (1961).16 Wyman, J., Jr., J. Biol. Chem., 127, 581 (1939).BREAKDOWN OF MESSENGER RNA DURING IN VITRO AMINOACID INCORPORATION INTO PROTEINSBY A. TISSIPRES* AND J. D. WATSONTHE BIOLOGICAL LABORATORIES, HARVARD UNIVERSITYCommunicated by John T. Edsall, April 3, 1962Messenger RNA (mRNA) which carries information for the synthesis of specificproteins from DNA to ribosomes, unlike ribosomal and transfer RNAs, is metabol-ically unstable.'-3 Thus a single mRNA molecule, attached to a ribosome, servesto make possibly one, or in any case a limited number of protein molecules. It isthen broken down, while new mRNA molecules, made on DNA templates, functionfor the synthesis of new proteins.There are now indications that mRNA is unstable in cell-free extracts and isbroken down to acid soluble fragments.4 5 In the work presented here, the condi-tions for this in vitro breakdown were studied. The results show that for mRNAbreakdown to occur, both ribosomes and supernatant are necessary. Moreover therate of breakdown is greatly increased by the addition of ATP and an ATP generat-ing system.Material and Methods.-1. C14 uracil labeling of mRNA: The pyrimidine requiring strain ofE. coli B 148 was cultivated, and the C14 uracil pulse labeling of T2 mRNA were done, essentiallyas previously described,5 with the exception that the cells were not starved for uracil before label-ing. Growth was followed by optical density measurements, and C14 uracil was added shortlybefore exhaustion of the unlabeled uracil present in the medium (4 ,ug per ml, allowing growthto about 5 X 108 cells per ml). The cells, washed twice in 0.005 M Tris-HCl pH 7.3 and 0.001 MMg++, were kept frozen.2. Preparation of cell-free extracts, ribosomes and supernatant: E. coli B was cultivated andwashed, and the crude cell-free extract was made by grinding with alumina and extracting with 3volumes of 0.005 M Tris-HCl pH 7.3 and 0.01 M Mg++, containing 5 ug deoxyribonuclease(I)Nase) per ml as described elsewhere.6 The washed cells could be kept frozen. In some experi-ments the extracts were fractionated into ribosomes and supernatant by 2 hr centrifugation at100,000g. The ribosomes were washed twice in at least twice the original volume of the extract ofTris-0.01 M Mg++ mixture. The upper 2/3 of the supernatant was recentrifuged for 2 hr at

Reversible Changes in the NEM-reactive--SH Groups of Hemoglobin on Oxygenation-deoxygenation

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Reversible Changes in the NEM-reactive--SH Groups of Hemoglobin on Oxygenation-deoxygenation

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