6I2.I27.3:6I2I.III.I H20 =H2CO reaction in blood. Their experiments gave strong. appreciable amounts of:

Transcription

1 68 6I2.I27.3:6I2I.III.I THE DIRECT CHEMICAL ESTIMATION OF CARBAMINO COMPOUNDS OF CO2 WITH HAMOGLOBIN. BY J. K. W. FERGUSON' AND F. J. W. ROUGHTON. (From the Physiological Laboratory, Cambridge.) (Received August 6, 1934.) INTRODUCTION. THE "bound" C02 of blood is defined as the difference between (a) The total C02 which can be liberated by treatment with acid and vacuum extraction, and (b) The C02 present in simple physical solution. The bound C02 certainly consists, in large measure, of free bicarbonate ions, but in addition there may, under physiological conditions, be appreciable amounts of: (i) C02 bound to haemoglobin in the form of carbamino compounds, as suggested by Henriques [1928]; (ii) C02 and/or HCO3 bound to the blood proteins in other forms. Meldrum and Roughton [1933] have recently described experiments on the rate of uptake of C02 by blood, in which the usual rapid formation of bicarbonate, via the reactions C02 + H H +HCO3, was inhibited by addition of 01M cyanide. The action of the latter is to poison the enzyme, carbonic anhydrase, which normally catalyses the 002 +H20 =H2CO reaction in blood. Their experiments gave strong support to Henriques' suggestion that C02 can combine rapidly, reversibly and in significant amounts with the haemoglobin of the blood to form compounds of a carbamino type according to the scheme: Hb NH2 + C02 Hb NHCOOH = Hb NHCOO + H. The number of free NH2 groups in the haemoglobin is known to increase with ph, and in agreement with this the amount of compound formed was found to be greater as the ph was made more alkaline. It was also 1 National Research Council Fellow.

2 H.EMOGLOBIN CARBAMINO COMPOUNDS. increased by lowering the temperature, and by substituting reduced hlemoglobin for oxyhaemoglobin. The objectives of the present papers were three in number: (i) To replace, if possible, the indirect method of Meldrum and Rough ton by a direct chemical method, which should be able to separate the carbamino-bound C02 from the bicarbonate and to estimate the former by direct chemical analysis. If this could be done, the actual existence of carbamino-bound C02 in the blood could scarcely be doubted. (ii) To estimate the amount of carbamino C02 under physiological conditions of temperature and ph, and thence to assess the importance of such compounds in the actual transport of C02 in vivo. The previous method of Meldrum and Roughton is ill suited to this important problem. (iii) To study further the effect of various factors upon the formation of carbamino compounds between C02 and hsemoglobin, and to discuss the relation of such compounds to the recent work of others on the state of C02 in blood. Of these (i) is dealt with in the present paper, whilst (ii) and (iii) will be treated in the adjoining paper [Ferguson and Roughton, 1934b]. A preliminary communication on these matters has already been given in this Journal [Ferguson and Roughton, 1934a]. Meldrum and Roughton [1933] made a start on a direct chemical method, by trying out with haemoglobin solutions the procedure which Faurholt [1925] has used so successfully for estimating the carbamino compounds of ammonia, methylamine, glycine, etc. This consists in adding sufficient NaOH to bring the reaction to a ph of 12-13, thus converting the whole of the H2C03 and the HC03 of the solution to the form of C03. The C03 is then precipitated by addition of excess of BaCl2. The barium salts of the carbamino compounds are, however, soluble, and at this very alkaline ph are sufficiently stable to be separated by centrifuging from the BaCO3 precipitate. From the C02 content of the supernatant fluid, as estimated either by titration with acid or by gasometric analysis, the carbamino content of the original solution can be calculated. The preliminary results of Meldrum and Roughton were such as to encourage us to develop the method into a quantitative one. In the present paper Section I describes the technique and special precautions necessary for the successful application of the method to 69

3 70 J. K. W. FERGUSON AND F. J. W. ROUGHTON. purified hemoglobin solutions, whilst Section II gives the evidence that the compound estimated in Section I is a direct carbamino compound of C02 with hamoglobin. It may be well to emphasize at the outset that the method described below only claims to measure the C02 combined with heemoglobin in the carbamino form and does not measure the C02 or bicarbonate bound to the blood proteins in other forms, if any. SECTION I. EXPERIMENTAL METHODS AND CONTROLS. Introduction. In order to appraise the reliability of our method, it will be well first to describe. shortly the factors concerned in the rate of dissociation of carbamino compounds, since these have largely governed the actual development of the technique. Faurholt has shown that the simpler carbamino compounds (e.g. of ammonia, glycine, etc.) dissociate according to the scheme PNHCOOHE = PNH2+C02,... (1) C02+OH HCO3,... (2) HCO3+OH C03+H20,... (3) all three reactions proceeding from left to right. The rate of dissociation becomes slower as the ph is made more alkaline-thus in the case of glycine at 18 C. the time for 20 p.c. dissociation at ph 12 is c. 6 min., at ph 13 c. 60 min. and at ph 14 c. 600 min. At 0 C. the times are about 20 times longer. In this very slow ph range, i.e. above c. ph 12, the overall rate of dissociation seems to be limited by some intrinsic property' of the carbamino compound, but at lower ph's the overall rate is conditioned by the rate of reaction (2), i.e. the rate at which C02, one of the products of the actual dissociation, is removed from the sphere of action. It is reasonable to suppose that the same principles would apply to the dissociation of the carbamino compound of a complex protein like haemoglobin. In haemoglobin solutions, even if well purified, there is, however, a complication not met with in the simpler cases studied by Faurholt, namely the presence of abundant amounts of the enzyme, carbonic anhydrase. This enzyme speeds up reaction (2) enormously, and must 1 A simple explanation of this may be suggested, namely that CO can only dissociate from the carbamino molecule, PNHCOOH, and not from its ion, PNHCOO. If so, then the rate of the first stage in the dissociation of CO should be proportional to [PNHCOOH], and should therefore, as the ph is made more alkaline, decrease until finally it becomes slower than the rate of reaction (2).

4 HEMOGLOBIN CARBAMINO COMPOUNDS. thus correspondingly speed up the overall rate of dissociation of the carbamino compound at ph < 12. If no loss of the latter is to occur in the process of estimation, it is absolutely necessary, not only to add sufficient alkali to bring the ph to the region where the overall rate of dissociation is independent of reaction (2), but also to mix the alkali with the hamoglobin so thoroughly and rapidly that there is not time for appreciable carbamino dissociation to occur in local patches of the fluid, where the haemoglobin is in excess during the actual nixmg process (where, in consequence, the ph would be less than the critical 71 Outer centrifuge cup I Inner centrifuge cup ~~~Water-bath ~~~~Thermostat Movable platform Fig. 1. value of, say, 12). On this account it was impracticable to use ordinary methods of mixing the reagents together in beakers, but it was necessary instead to adopt the arrangement now to be described. Experimental method. Three 5 c.c. syringes of equal bore are suspended vertically from a rigid metal bar as shown in Fig. 1. Below them is a movable platform, which can be raised and lowered by turning a crank. When the platform is raised it pushes up the plungers of the syringes thereby expelling their contents simultaneously in exactly equal quantities. Pressure tubing of 2 mm. bore connects the tip of each syringe to a Hartridge-Roughton mixing chamber (loaned by G. A. Millikan). It consists essentially of a 1 mm. four-way stopcock with the barrel bored in such a way that the three incoming reagents are mixed in the minimum

5 72 J. K. W. FERGUSON AND F. J. W. ROUGHTON. time, and the mixture expelled through the fourth arm of the stopcock or effluent tube. In an ordinary experiment the contents of syringes (a), (b), (c) are as follows: (a) The hemoglobin solution to be analysed for carbamino C02. (b) 7 p.c. BaCl2 in NaOH solution of suitable strength. The factors on which the latter depends will be discussed later. (c) Distilled water in early stages of the work (so as to make the analysis comparable with the controls to be described later). Lately, however, for reasons to be discussed below, bicarbonate solutions were used instead. The platform, syringes and mixing chamber are immersed in a water thermostat usually at C. The tube leading from the mixing chamber protrudes from the water bath, and at the appropriate time a 15 c.c. centrifuge tube surrounded by ice is held in a suitable position to catch the effluent mixture. When temperature equilibrium has been attained (15 min.) the platform is elevated by turning the crank. The three reagents are simultaneously expelled, mixed in the chamber in less than 0 01 sec., and shot out through the effluent tube. The syringes are emptied in about 5 sec. The first 2-3 c.c. of effluent are discarded. The remainder is caught in the centrifuge tube and immediately covered with liquid paraffin. The mixture is centrifuged in ice for 15 win. Ice temperature helps to reduce the rate of dissociation of carbamino compounds. A 5 c.c. sample of the supernatant fluid is then analysed for C02 by the well-known van Slyke technique. Preparation of haemoglobin solution. Nearly all our recorded experiments have been done on hawmoglobin solutions, purified by Adair's method, in which washed red cells are laked with ether, the stroma proteins precipitated with NaCl and removed by centrifuging, and the NaCl and ether subsequently removed by dialysis through a collodion membrane. The solutions were preserved at - 10 C. and small quantities thawed out on the same day as they were to be used. Some experiments have been done on haemoglobin solution prepared by laking washed cells by freezing and removing the stromata by adsorption with Marshall and Welker's [1913] alumina cream. This preparation was abandoned because the yield of carbamino compound was only about two-thirds of that obtained with Ada ir's haemoglobin. It is possible that some of the reactive NH2 groups were blocked by adsorbed alumina. Strength of alkali added. The addition of a large excess of alkali to a heemoglobin solution to secure the stability of the carbamino compound

6 HAMOGLOBIN CARBAMINO COMPOUNDS. was not feasible, because such treatment would quickly denature the htemoglobin. It is not self-evident what effect such dehaturation would have on the yield of carbamino compound as estimated by this method. It would probably be diminished, but in any case serious doubts would be cast on the validity of the results. Indeed Henriques [1933] CB 4 4) 3 o J Reduced,H o 02HB 2-~~~~~~~~ 44.ON ~Bicarbonate controls Normality of added alkali (in syringe (b)) Fig. 2. Effect of strength of alkali added on the yield of unprecipitated C02 in bicarbonate controls and experiments on oxygenated and reduced Hb, at constant Pco2 and C02 content. abandoned the method for this reason. On the other hand, no method of adjusting the alkalinity to a definite ph was readily available, nor indeed was the required ph known. Instead, various amounts of alkali were added to aliquot portions of a htemoglobin solution in equilibrium with a definite tension of 002. The yield of carbamino compound was plotted against the strength of alkali added. Two experiments are illustrated in Fig. 2.

7 74 J. K. W. FERGUSON AND F. J. W. ROUGHTON. At low strengths of added alkali large amounts of C02 were found in the supernatant fluid, presumably because insufficient alkali had been added to change all the bicarbonate to carbonate. The unchanged portion could not be precipitated by the BaCl2 since barium bicarbonate is soluble. The curve then passes through a minimum where sufficient alkali has been added to turn all the bicarbonate into carbonate but not enough to stabilize the carbamino compound. As more alkali is added a plateau is reached, which, however, rises slightly because the rate of dissociation is still falling perceptibly with rising ph and/or because the mixing in the chamber is not rapid and thorough enough. In controls, with an alkalinity corresponding to the middle of the plateau, the centrifuging was prolonged beyond the usual 15-min. period. These showed that during the first 15 min. of centrifuging an average dissociation of c. 8 p.c. must have occurred. On this basis it is found that the higher yield of carbamino compound at the top of the plateau can be accounted for by the smaller amount of dissociation at the more alkaline ph; it may therefore be taken that the efficiency of the mixing, per se, is adequate, and that the danger, alluded to on p. 71, of appreciable carbamino dissociation in local patches of the fluid during the mixi g process has been circumvented. In our usual experiments the [NaOH] used corresponded to the middle of the plateau, and to the observed carbamino content a correction of 8 p.c. has accordingly been added. The actual concentration of NaOH mols per litre, to be used in syringe (b), was given by the formula [NaOH] = 10 [Hb] + [CO2], where [Hb] = oxygen capacity of the haemoglobin solution (in syringe (a)) expressed in mols/litre, [CO2] =total C02 content of the haemoglobin solution (in syringe (a)) expressed in mols/litre. The chief difficulties met with experimentally were (i) haematin formation, (ii) the effect of the proteins in protecting the BaCO3 precipitate from complete separation in the centrifuge, (iii) uncertainty as to the fate of the dissolved C02 originally present in the haemoglobin solution. These are discussed seriatim. Haematinformation. The difficulty encounteredcbyhenriques [1933] seems to be obviated by the use of ox haemoglobin, which is known to be particularly resistant to alkaline denaturation. Only when the largest amounts of alkali were added was there any appearance of haematin formation. Thus by using an alkalinity corresponding to the middle of plateaux shown in Fig. 2 a standard yield of carbamino compound was

8 H.EMOGLOBIN CARBAMINO COMPOUNDS assured, and trouble from haematin formation avoided. Our controls on this point were as follows: (i) In three experiments the denaturation or loss of h,emoglobin was estimated by measuring the 02 capacity of the alkaline supernatant fluid. The loss was found to amount on the average to 5 p.c. of the total. A 2-3 p.c. loss can be accounted for by the carrying down of haemoglobin by the BaCO3 precipitate. This was estimated by redissolving the washed precipitate in acid and measuring its haemoglobin content colorimetrically. (ii) In three experiments an attempt was made to detect denatured hwemoglobin spectroscopically. Sodium hydrosulphite was added to the supernatant fluid to reduce all kathaemoglobin to haemochromogen-the spectrum of the latter is detectable in concentrations of 2-3 p.c. in reduced hsemoglobin. No sign of its spectrum was seen, indicating that less than 2 p.c. of hwemochromogen was present in the supernatant. Assuming that 2 p.c. hwemochromogen is formed the greater part of the loss of active haemoglobin can be accounted for. Bicarbonate controls. It was important to ascertain whether or not haemoglobin prevented in any way the complete sedimentation of the BaCO3 on centrifuging. To control this point the apparatus was used exactly as before, except that the syringes were filled with the following solutions: Syringe (a): haemoglobin solution rendered almost free of C02 (i.e. < 0 4 c.c./100 c.c.) by adjustment of its ph to c. 6-5 with acetic acid or HCI, and by repeated evacuation. Syringe (b): 7 p.c. BaC0 in alkali. Syringe (c): a solution of sodium bicarbonate of C02 content varying from 4 v.p.c.1 to 300 v.p.c. The procedure was exactly that of an ordinary experiment. Analysis of the supernatant fluid in the case of two different hsemoglobin preparations gave the series of results plotted in Fig. 3 (temperature 38 C.). At low concentrations of added bicarbonate, almost the whole of the added bicarbonate fails to be precipitated,' but as the concentration is raised the proportion precipitated rapidly increases until at a bicarbonate content=50 v.p.c. C02 only about 0'5 v.p.c. C02 remains in the supernatant fluid. The following factors were without influence: (i) oxygenation or reduction of the heemoglobin, (ii) adjustment of the initial ph of the 1 For convenience the total C02, the dissolved C02, the bicarbonate and the carbamino concentrations are, in these two papers, all measured in the customary physiological units of volumes p.c. (v.p.c.). This means the number of c.c. of gaseous 002 [N.T.P.] per 100 c.c. of solution, to which the concentration of the substance in question is chemically equivalent.

9 76 J. K. W. FERGUSON AND F..J. W. ROUGHTON. heemoglobin solution to c. 8-0 instead of to c. 6-5, (iii) presence of traces of octyl alcohol. This interesting and somewhat unexpected relationship is almost certainly to be explained by the protective action of heemoglobin on the precipitation of BaCO3. At low concentrations of NaHCO3 the precipitate *Plasma 0c) 04 c; Go 0 (2 PI V * Serum Laked blood so Total C02 to be precipitated, i.e. [HCO3] expressed as v.p.c. C02, in syringe (c) Fig. 3. The effect of total bicarbonate concentration on the proportion of it precipitated as BaCO3 in the presence of Hb. x =reduced Rb * =reucdhb +=;reduced Rb of first sample of houmoglobin. of second sample of hlemoglobin. Labelled points enable comparison to be made of the precipitability of BaCO3 in various protein containing solutions. of BaCO3 should grow relatively slowly and there would be time for heemoglobin to be absorbed over the surface of the incipient BaCO3 nuclei, and thus prevent them growing to a sufficient size to come down in the centrifuging; at higher concentrations of NaHCO3 the BaCO3 precipitate should, however, grow faster and have time to reach a centrifugable size before it becomes covered in whole (or perhaps a part is only possible in these circumstances) with a protective layer of heemoglobin.

10 HAiMOGLOBIN CARBAMINO COMPOUNDS. The marked discrepancy between the two curves plotted in Fig. 3 shows that individual hemoglobins differ quite markedly in their protective power. Similar experiments with laked blood, plasma and serum show that the other proteins of the blood are far more effective than haemoglobin in obstructing the precipitation of BaCO3-isolated examples of this are also shown in Fig. 3. This, however, is not a serious drawback from the physiological point of view, since experiments by the method of Meldrum and Roughton [1933] suggest that the plasma proteins do not, under physiological conditions, bind appreciable amounts of C02 in the carbamino form. For this reason we have not yet tried to extend our present technique beyond the case of purified heemoglobin solutions. We have in the main studied the carbamino content of haemoglobin solutions containing bicarbonate = v.p.c., since (i) the blank corrections for unprecipitated bicarbonate are according to Fig. 3 relatively slight, (ii) this is the range of concentration which occurs under physiological conditions in the blood. In an actual carbamino-co2 estimation conditions are not quite the same as in the preceding controls, for in the former the bicarbonate which it is wished to precipitate is "molecularly" mixed with the haemoglobin, before it is driven into the mixing chamber, and so the protective action of the haomoglobin might be somewhat different from what it is in the controls, wherein the bicarbonate is placed in a separate syringe from the hawmoglobin. To meet this point, a further series of rather complex controls was carried out. In the first experiment of one control: Syringe (a) contained 10 mm. reduced haemoglobin, adjusted to ph c. 8-0 by addition of C02-free NaOH, and then NaHCO3 added thereto so as to make the solution=32-3 v.p.c. C02. On standing a small amount of dissolved C02 will form (of the order of 0 3 v.p.c.) and probably a larger amount of carbamino compound (of the order of 2-5 v.p.c. C02). Syringe (b) contained the usual BaCl2-NaOH mixture. Syringe (c) contained distilled water. Analysis of the supernatant fluid showed that of the 32-3 v.p.c. C02 in solution (a) 5-73 v.p.c. were unprecipitated. Therefore 5.73 v.p.c. = unprecipitated bicarbonate + carbamino C02 of solution (a). In the second experiment of the same control: syringes (a), (b) contained the same solutions as in the first experiment of the control, syringe (c) contained NaHCO3 solution= 100 v.p.c. C02. 77

11 78 J. K. W. FERGUSON AND F. J. W. ROUGHTON. On the basis of Fig. 3 the effect of this should be to precipitate the whole of the bicarbonate of solution (a), apart from a small blank= 0-2 v.p.c. CO2 which occurs even at high bicarbonate concentrations. Analysis showed that in this case 3*93 v.p.c. C02 of solution (a) was unprecipitated. Subtracting from this a blank of 0-2 v.p.c. we get a residue of 3-73 v.p.c. C02 which must be due to the carbamino0c2 of solution (a). The unprecipitated C02 of solution (a) thus amounts to *73 = 2 v.p.c. C02. In the third experiment of the control: Syringe (a) contained 10 mm. reduced Hb free of C02 at ph 8&0 with no added bicarbonate. Syringe (b) contained the usual BaCl2-NaOH mixture. Syringe (c) contained NaHCO3 solution= 30 v.p.c. C02. The unprecipitated C02 in the actual supernatant fluid was found to be 0 74 v.p.c. Since each syringe contributed exactly one-third of the whole mixed fluid this means that 3 x 074, i.e. 2x22 v.p.c. C02 of solution (c) was unprecipitated. This agrees so closely with the previous value of 2*0 v.p.c. as calculated from the first and second experiments of the control that it seems fair to conclude that the precipitation of BaCO3 from a solution containing NaHCO3= 30 v.p.c. C02 is the same whether the bicarbonate is molecularly mixed with the hwemoglobin before or after being driven into the mixing chamber. Five controls of this kind are listed in Table I. TABLE I. Condition and Bicarbonate in CO2 unprecipitated v.p.c. concentration syringe (a) t A of hsmoglobin in v.p.c. C02 Exp. 1 - Exp. 2 Exp mm. reduced = mm. oxygenated = mm. reduced = mm. oxygenated *05 2*06 10 mm. reduced = Temperature 37 C. In the first four experiments of Table I the agreement is within experimental error. The technique therefore seems sound, both for oxyand reduced haemoglobin, when the bicarbonate concentration is greater than 30 v.p.c. C02. In the last experiment, however, there is a serious discrepancy, which is perhaps not unexpected, since the bicarbonate concentration, viz. 8 v.p.c. C02, lies in the low range, where the effect of haemoglobin in preventing the sedimentation of BaCO3 precipitates is

12 HEMOGLOBIN CARBAMINO COMPOUNDS. relatively very serious. On this account we do not place any trust in our method, as so far developed, for concentrations of bicarbonate less than 30 v.p.c. C02, though luckily the method seems to be sound, as regards this particular matter, in the physiological range of bicarbonate concentration. From these results it is clear that, unless the bicarbonate concentration in the haemoglobin solution exceeds 100 v.p.c. C02, a compensatory amount of NaHCO3 should be placed in syringe (c). This has been done in our recent experiments, and we intend to do so always in the future. Some of our earlier experiments were carried out without proper attention to this factor-except in cases where approximate allowance was possible the data so obtained have been discarded. Two experiments, which were in all respects similar to the bicarbonate controls except that distilled water was used in syringe (a) in place of haemoglobin, showed only minimal traces of C02 in the final supernatant fluid-viz and 0-08 v.p.c. respectively. C02 controls. The experiments described under the heading "Bicarbonate controls " fall short of being complete in one important respect, namely that no appreciable dissolved C02 is involved. When a C02- equilibrated hawmoglobin solution is suddenly made alkaline, the dissolved C02 may not all be converted into CO, even though this reaction would be catalysed by the abundant amounts of the enzyme, carbonic anhydrase, present in all samples of Hb prepared by Adair's method. A part of the dissolved C02 may combine with heemoglobin to form new carbamino compounds which would, unless controlled for, be reckoned as preformed carbamino C02. It will be remembered that hoemoglobin, like other NH2 compounds, forms carbamino compounds more readily at alkaline than at neutral ph [Meldrum and Roughton, 1933]. This source of error was investigated in the following way: Syringe (a) contained 10 mm. oxyha3moglobin free of C02 ph c Syringe (b) contained the usual BaCl2-NaOH mixture. Syringe (c) contained NaHCO3 solution = 100 v.p.c. C02, equilibrated with a C02 pressure of 75 mm. Hg at 370 C. The supernatant fluid contained 0-38 v.p.c. unprecipitated C02. Allowing the usual small blank for unprecipitated bicarbonate this would mean that 17*5 p.c. of the dissolved C02 in solution (c), i.e v.p.c. C02, was unprecipitated, presumably because it formed carbamino compounds when the haemoglobin was mixed with alkali. Roughly the same result 79

13 80 J. K. W. FERGUSON AND F. J. W. ROUGHTON. was found when reduced haemoglobin was used in place of oxyheemoglobin, and when the initial ph was c. 8&0 instead of c Experiments of this type are also not quite perfect, in that the dissolved C02 was not in molecular contact with the heemoglobin b e f o re the solutions were driven into the mixing chamber. To meet this point roughly: Syringe (a) contained 10 mm. oxyhaemoglobin equilibrated with C02 pressure=85 mm. Hg total C02=31'5 v.p.c., ph=c. 6-8, dissolved C02 practically the same as in the previous experiment. Under these conditions oxyhaemoglobin only binds a very small amount of carbamino C02. Syringe (b) contained the usual BaCl2-NaOH mixture. Syringe (c) contained NaHCO3 solution = 100 v.p.c. C02. Analysis of the supernatant fluid showed, that after the usual blank corrections had been made, 1X56 v.p.c. C02 of solution (a) was unprecipitated. The latter=the carbamino C02 originally present in (a)+a fraction of the dissolved C02 originally present in (a). Unfortunately we had no means of knowing how much preformed carbamino C02 was present in solution (a), but it is not unreasonable, from our general results, to assume that it was sufficient to account for the discrepancy between the value of 1X56 v.p.c. C02 in the present experiment and the value of 0 93 v.p.c. C02 in the previous experiment, i.e. that the preformed carbamino C02 in solution (a) amounted to c. 0x6 v.p.c. C02. We therefore feel on fairly safe ground in holding that the behaviour of dissolved C02, when present in the haemoglobin syringe, does not differ materially from its behaviour when present in one of the other syringes, provided that the heemoglobin solution does not contain less bicarbonate than 30 v.p.c. C02, and that the total bicarbonate supplied by the three syringes is adequate (vide subsection on " Bicarbonate controls "). In actual estimations carried out under these conditions we propose to allow for the effect of dissolved C02 by subtracting from the unprecipitated C02 a blank= 15 p.c. of the dissolved C02 initially present in the haemoglobin solutions. In much of our earlier work on dissolved C02 controls we failed, through ignorance, to have enough bicarbonate in the haemoglobin and/or other solutions. In these cases p.c. of the dissolved C02 was unprecipitated: a large part of this was probably to be accounted for, not by new carbamiino formation when the solution was made suddenly alkaline, but by imperfect precipitation of BaCO3. No signifi-

14 HAMOGLOBIN CARBAMINO COMPOUNDS. cant difference, however, was found between oxy- and reduced hasmoglobin, even under these unfavourable circumstances. Recoverability of added carbamino C02 (in the form of ammonium carbamate). In this test the contents of the syringes were: (a) Hb solution almost free of C02 ( < 0.5 v.p.c.). (b) 7 p.c. BaCl2 in NaOH. (c) A solution of ammonium carbamate, C02 content = 114 v.p.c., prepared by shaking a solution of NH3 in NaOH with C02 at 00C., and removing any carbonate so formed (as distinct from carbamate) by precipitation with excess of BaC12. In the mixed fluid the carbamino C02 should have been 38 v.p.c.; the amount actually recovered in the supernatant fluid was a shade more, viz v.p.c. Summary of reagent strengths and corrections. For convenience the solutions in an ordinary experiment and in the two most important controls are summarized in Table II. TABLE II. Syringes,~~~~~~A Exp. (a) (b) (c) Ordinary Hb solution equilibrated 7 p.c. BaC12 in NaOH NaHCOs solution Bicarbonate control CO2 control with C02 Hb solution C02 free oil The corrections to be applied to experimental results may also be summarized as follows: (I) For errors tending to high results: Approximate correction (1) Dissolved CO2-carbamino compound 15 p.c. of dissolved CO (2) Incomplete precipitation of CO, by Ba v.p.c. C02 in supernatant fluid (depending on sample of Rb) (II) For errors tending to low results: (1) Los of Hb in BaCOs precipitation 2-3 p.c. of carbamino C02 (2) Dissociation of carbamino compound 8 p.c. of carbamino C00 during centrifuging In most cases the sum of all these corrections did not amount to more than 0 5 v.p.c. C02. The calculation of the results in a typical experiment. Oxygen capacity of heemoglobin solution= 22 v.p.c. Reduced Hb solution equilibrated at 380 C. with pco2 = 32.6 mm. Hg. Total C02= 31-0 v.p.c. in equilibrated solution. C02 content of supernatant fluid = 0 97 v.p.c. C02, i.e x 3 = 2*9 v.p.c. C02 in terms of Hb solution in syringe (a). PH. LXXXIII. 6 81

15 82 J. K. W. FERGUSON AND F. J. W. ROUGHTON. Corrections for (i) Bicarbonate control-subtract 0-2 v.p.c. (ii) Dissociation of carbamino C02 during centrifuging, and carrying down of Rb by BaCO3 precipitate-add 10 p.c. of ( ), i.e v.p.c. (iii) Dissolved C02 which- turns into carbamino C02 on making alkaline-subtract 15 p.c. of dissolved C02 content of Hb solution in syringe (a), i.e. 15 p.c. of 2-07 = 0-31 v.p.c. Net preformed carbamino C02 = = 2-65±0-4 v.p.c. SECTION II. EVIDENCE FOR THE NATURE OF THE C02 COMPOUND ESTIMATION IN SECTION I. A. Evidence that the C02 found in the supernatant fluid is bound to hcmoglobin. The unprecipitated C02 (apart from that allowed for by the various blank corrections) must be bound to some compound in the haemoglobin solution, otherwise it could not escape precipitation by barium. In haemoglobin solutions, after dialysis by Adair's method, the only possible substances which could bind C02 would be either hemoglobin, or one or more of the other colloids (including enzymes) of the red blood corpuscles, which are not removed in the precipitation process. Three arguments point to the binder being, in the main, haemoglobin: (i) There is no other colloid, present in sufficient quantity to be likely to bind the amounts of C02, which is actually observed to be bound. (ii) The amount bound, at constant ph, temperature and [CO2, is roughly proportional to the hwemoglobin concentration. This is shown by the three experiments at different concentrations of reduced hemoglobhi listed in Table III. TABLE III. Effect of himoglobin concentration. Hb in Temp. Approx. Carbamino mm. carbamino CO. mm./litre C. pco2 ph CO2 v.p.c. mm. Hb ^O ' *42 Average 0-48 (iii) At physiological ph, temperature and C02 pressure, reduced htemoglobin solution is found to bind appreciable quantities of C02, but oxygenation of the same solution reduces the amount bound, under physiological conditions, practically to zero. In purified haemoglobin

16 HAiMOGLOBIN CARBAMINO COMPOUNDS. solutions there is no substance, other than hwemoglobin, which is known to be affected by oxygenation. On these three grounds, but especially (iii), it seems reasonable to take it that the unprecipitated C02 is, in the main, a ha3moglobin-co2 compound. B. Evidence that the hcwmoglobin-c02 compound is a carbamino compound. (i) This is strongly suggested by the behaviour shown by the compound in the course of its separation and estimation, as described in Section I. (a) The compound is not precipitated by BaCl2 at alkaline ph. (b) The compound dissociates less rapidly as the ph is made more alkaline, and is relatively stable at 00 C. above ph c. 12. (c) The compound is rendered less stable by rise of temperature. In all these three respects the compound behaves in a manner similar to the carbamino compounds of the simpler compounds-ammonia, glycine, etc.-as studied by Faurholt. (ii) If the C02 compound is a carbamino one, acidification, according to Faurholt, should cause the direct splitting off of C02 molecules. This was tested as follows: 2-0 c.c. of supernatant fluid from an experiment, as in Section I, were treated with M/7 KCN (so as to poison the carbonic anhydrase, which is still present and active in the supernatant fluid) and then placed in an 8 c.c. vessel attached to a manometer. By means of a small syringe, 0-5 c.c. of M phosphate buffer, ph c. 5'6, was suddenly squirted into the vessel, which was then shaken violently for 10 min. The course of C02 evolution, as observed manometrically, is shown in Fig. 4, A. The large output of C02 during the first 15 sec. is due to the fact that the C02 molecules are split off more rapidly from the Hb-CO2 compound than they can be hydrated (by the reactions C02 + H20 --H2CO3 = H + HOG3). Those C02 molecules which have not time to be hydrated in the 15 sec. therefore escape into the gas phase, only to be reabsorbed again during the subsequent shaking on account of the slow hydration reaction. If, however, the cyanide is omitted so that the carbonic anhydrase is fully active, the hydration reactions should be rapid enough to take up the C02 as fast as it is formed, so that in this case there should be no "overshooting." Fig. 4, B, shows that this is indeed the case. The final ph in Fig. 4, B, was c. 1 ph unit more acid than in Fig. 4, A: this explain why the end point is different

17 84 J. K. W. FERGUSON AND F. J. W. ROUGHTON. Fig. 5 shows a similar pair of experiments with a solution of ammonium carbamate (free from carbonate), in place of the Hb-CO2 solution. In the first of these (Fig. 5, A) the ammonium carbamate (which contains no carbonic anhydrase) was simply mixed with phosphate buffer, so that the result as regards "overshooting" should have been, and indeed was, similar to that of Fig. 4, A. In the second one (Fig. 5, B) Seconds Fig. 4. Course of CO2 evolution when alkaline carbamino containing hasmoglobin mixed suddenly with phosphate buffer ph 5*6. A, with M/7 KON added to Hb, i.e. carbonic anhydrase therefore inactive; B, no KCN added, i.e. carbonic anhydrae active. Final ph in A more alkaline than in B. Temperature 160 C. carbonic anhydrase was deliberately added and the "overshooting" effect is now eliminated as in Fig. 4, B. In the two experiments shown in Fig. 5 the final ph's were the' same, and thus the end points were also identical. This same kind of test has been applied successfully by Krebs and Roughton [1934] to other reactions in which C02 is directly produced. Its success, in the present instance, shows that a large proportion, at any rate, of the C02 bound in the supernatant fluid is bound as CO2, and not as Hco3 or C03. Had the latter been the case, acidification

18 HAEMOGLOBIN CARBAMINO COMPOUNDS. should have led to a smaller evolution of C02 during the first 15 sec., when cyanide is present so as to poison the carbonic anhydrase, than if the latter is fully active. (iii) The effect of ph and temperature on the formation of the haemoglobin-co2 compound agrees closely with expectation (based on Faurholt's work) if the compound is a carbamino one. Our study of the effect of these factors and also of the effect of oxygenation is described Seconds Fvig. B. Course of C02 evolution when ammonium carbamate (NH2COONH4) mixed suddenly with phosphate buffer (ph 5 8). A, without added carbonic anhydrase; B, with added carbonic anhydrase. Temperature 0 C. Final ph c in more detail and discussed in the adjoining paper [Ferguson and Roughton, 1934b]. The results given there also confirm those obtained by Meldru mand Ro ug ht o n[1933] bytheir quite different method, which goes to show that the same type of compound was being dealt with in their case, as in ours. These various lines of evidence converge to show that the C02 compound found in the supernatant fluid, in the estimations of Section I, ist a carbamino compound of C02 with haemoglobin. This will be accepted by us, for the present, as a working hypothesis, and, as such, made use Of in our acdjoinig paper.

19 86 J. K. W. FERGUSON AND F. J. W. ROUGHTON. SUMMARY. 1. A method is described for precipitating, as barium carbonate, practically the whole of the bicarbonate, and most of the dissolved C02 present in haemoglobin solutions, which have been equilibrated with gas mixtures containing CO2. It consists in mixing suddenly in a Hartri dg e- Roughton mixing apparatus, (a) the solution of haemoglobin containing C02, (b) 6 p.c. BaCl2 dissolved in a strength of NaOH, (c) NaHCO3 solution of suitable strength. The fluid emerging from the mixing chamber is caught in a centrifuge tube, and at once centrifuged for 15 min. at about The BaCO3 precipitate is found in the centrifugate. 2. The supernatant fluid is analysed for unprecipitated C02 in the van Slyke gasometric apparatus. Appreciable amounts are often found: evidence is given that these are mainly due to preformed carbamino compounds of C02 with haemoglobin in the original heemoglobin solution. 3. In computing from the C02 content of the final supernatant fluid the actual amount of preformed carbamino C02 in the haemoglobin solutions, blank corrections have to be allowed for (a) the dissociation of the carbamino compounds during centrifuging, (b) hematin formation, (c) the carrying down of some of the haemoglobin with the BaCO3 precipitate, (d) the not quite complete precipitation of the bicarbonate, (e) the conversion of some of the dissolved C02 originally present in the haemoglobin into new carbamino compounds when the mixture is suddenly made alkaline. The overall effect of all these corrections is, however, slight compared with the amount of carbamino bound C02 often found in hawmoglobin solutions at 370 C. over the ph range The method is not at present applicable to plasma, serum, laked blood, or to purified haemoglobin solutions containing less bicarbonate than that corresponding to about 30 v.p.c. C02. REFERENCES. Faurholt, C. (1925). J. chim. Phy8. 22, 1. Ferguson, J. K. W. and Roughton, F. J. W. (1934a). J. Phy8iol. 81, 21P. Ferguson, J. K. W. and Roughton, F. J. W. (1934b). Ibid. 83, 87. Henriques, 0. M. (1928). Biochem. Z. 200, 1, 5, 10, 18, 22. Henriques, 0. M. (1933). Ibid. 260, 58. Krebs, H. A. and Roughton, F. J. W. (1934). Unpublished. Marshall, J. and Welker, W. H. (1913). J. Amer. chem. Soc. 35, 820. Meldrum, N. U. and Roughton, F. J. W. (1933). J. Phy8iol. 80, 143.