CONFERENCE ON HEMOGLOBIN 2-3 MAY 1957 (1958) / Chapter Skim
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From page 1... ...
Whatever the exact composition of the earth's primeval atmosphere, the reactions that would have occurred in that atmosphere under the influence of ultraviolet light and electric storms would have led to the formation of at least some of the amino acids. As to the porphyrins, there is ample evidence from deposits of great antiquity of their presence in nature many hundreds of millions of years ago, at least.
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From page 2... ...
Since we shall hear about the amino acid analysis of hemoglobin later from Dr. Stein, I will not try to discuss that here, except to remind you of oIle very striking feature, which has long been known—namely the large number of histidine residues, approximately 35 per hemoglobin molecule.
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From page 3... ...
, has far-reaching effects on the oxygen amenity of the hemoglobin molecule and on the interactions between the heme groups. Steinhardt and Zaiseri3 have shown that hemoglobin contains approximately 36 negatively charged groups, and apparently an equal number of positively charged groups, which in the native molecule are non-titratable with acids and bases, but become titratable upon acid, and perhaps also upon alkaline, denaturation.
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From page 4... ...
containing four binding sites, to each of which a ligand X may be attached. For our present purposes, X is oxygen of perhaps carbon monoxide.
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From page 5... ...
If the system shows positive interactions, the binding of one X facilitates the binding of the next, and art > x3 > ^~2 > Act. It has long been known that the interaction of hemoglobin with oxygen (or carbon monoxide ~ involves strong positive interactions; WymanS gave a searching discussion of the data available ten years ago.
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From page 6... ...
Positive interactions are shown by a positive, negative interactions by a negative slope. Thus the copper-imidazole systems show strong negative interactions, while the interactions in the zinc-imidazole systems are strongly positive.iS~~9 We have taken some of the beautiful data on hemoglobin-oxyger~ interactions from Lyster's thesis, kindly made available to us by Professor Roughton and Dr.
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From page 7... ...
Interactions of such intensity have suggested to more than one inquirer in recent years the conception that oxygenation (or combination with carbon monoxide, or oxidation to ferrihemoglobin ~ must lead to a rather drastic rearrangement of the total configuration of the molecule as a whole. Such thoughts were perhaps first expressed by St.
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From page 8... ...
There are striking and obvious changes in many physical properties of hemoglobin when it combines with oxygen or carbon monoxide. The changes in absorption spectra are too well known to need comment; likewise the change from the paramagnetic reduced hemoglobin ~ ferrohemoglobin ~ to the diamagnetic oxy- or carbonmonoxyhemoglobin, discovered by Pauling and Coryell, is most dramatic.
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From page 9... ...
This large and positive entropy difference is in the same direction, although smaller in magnitude, as that associated with protein denaturation. It suggests a change from a more ordered to a more disordered type of structure in the hemoglobin molecule, at least at certain stages in the oxygenation process, which is much more drastic than could readily be accounted for by local changes of bond character in the heme groups and closely adjoining amino acid residues.
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From page 10... ...
Recent studies of dielectric increments and dielectric dispersion by Takashima'~ and Takashima and Lumry~3 strongly reinforce the view that some rather far-reaching change in molecular configuration is associated with the oxygenation process. Figure 3 shows the dielectric increment and the relaxation 70 50 r,X 108 30 ·sc 10 b J ~ RELAXATION ~ DIEI~CTRIC INCREMENT 8.19 ~ _ 1 1 1 1 1 1 1 1 19 0 1 2 3 4 5 6 7 8 9 oxy O2 PART lAL PRE SSU RE FIG.
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From page 11... ...
I should like to elaborate on this problem further here, but any real discussion would take far too long. It is worth noting, however, that Theorell2` has made use of the now precisely known sequence of the amino acid residues adjoining the heme group in cytochrome c, to construct a model in which the imidazole group of the histidine residue in the peptide chain fits nicely on to the iron atom of the heme, if it is assumed that the peptide chain is coiled in an a-helix.
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From page 12... ...
H.: Amino acids formed in primitive atmospheres, Science 124: 935, 1956.
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From page 13... ...
Soc. See also Lumry and Takashima: Dielectric dispersion studies on the oxygenation of horse hemoglobin, Abstracts of 131st meeting, American Chemical Society, Miami, Florida, April 7-12, 1957, p.
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From page 14... ...
I will now get straight ahead with my main topic: the individual velocity constants in the chain of the four reactions of hemoglobin with oxygen, carbon rr~onoxide and nitric oxide. At Cambridge in 1948 wee only had very sparse k'~ Hb4 + X = Hb4X kl Karl l'l ——K1 _ Lo kl Hb4X + X=TIb4X, ~ - K
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From page 15... ...
Lyster.3 It will be noted in figure ~ that in the case of the oxygen reaction we use K symbols, in the case of carbon monoxide L symbols, and in the case of nitric oxide, not shown in the figure we use ~ symbols. Over sixty years ago, Haldane and Lorrain Smith first observed that light dissociated CO from hemoglobin.
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From page 16... ...
If the Rash is such as to split off the whole of the carbon monoxide, then there is only a relatively slow rate of recombination compared with that which occurs when a small fraction of the carbon monoxide is split offal The slope of the -0 1 1 30 >0 _ l ..
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From page 17... ...
The correction for the later reactions can be reduced to 3 per cent or less. These applications of flash photolysis have thus provided very telling methods of dissecting out two of the individual velocity constants of the chain of the carbon monoxide reactions with hemoglobin.
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From page 18... ...
Later, another and sharper example of that type of contradiction will be shown. The next two figures demonstrate the effects of PCMB and the reversal of the PC~B effect by the addition of glutathione on the two velocity constants.
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From page 19... ...
In a paper recently published in the B Series of the Royal Society of London,6 we have analysed the complete course of the kinetics of carbon monoxide and hemoglobin in terms of the four velocity constants, lit, lid, 1~3 and 1~4 in accordance with the equations: Hb4 + CO -~ Mb4CO, dt s q 1', U s I' qr — I' qs HO ~ CO ~ + CO - ` Hb4 ~ CO ~ I, dt Hb4(CO)
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From page 20... ...
what one would expect on the basis of the four velocity constants and what one observed in regard to the total amount of CO hemoglobin formed at various times from the beginning of the reaction. The full line curve in the top panel displays the observed course of the combination.
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From page 21... ...
Gibson and Roughton~ have shown (see figure 12) that the in dividual velocity constants of dissociation, as regards the first molecule
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From page 22... ...
FIG. 12.-Rel~tloD of actlvatloD ener~y to velocity constants.
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From page 23... ...
Figure 13 illustrates the method used by Gibson and RoughtonS two years ago for obtaining k4. A solution of carbon monoxide was mixed with oxyhemoglobin, which had been equilibrated with various pressures of oxygen ranging from a tenth of an atmosphere to a whole atmosphere, the carbon monoxide pressure being constant.
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From page 24... ...
The velocity constants of some of these later reactions are greater than k4 and as their influence comes more and more into play the calculated value of the overall dissociation constant correspondingly increases. The determination of lo, the velocity constant of the first carbon monoxide ~1 z30 o 920 cr 0 1 0 ~1 I-)
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From page 25... ...
Figure 17 shows a plot similar to figure 14 for a series of experiments in which CO hemoglobin in equilibrium with various pressures of CO was mixed with a buiEer containing some dissolved nitric oxide. Again a straight line is obtained but there is, in this case, an experimentally observed point on the vertical axis itself.l1 Such a point is obtained when a 100~7O CO hemoglobin solution in equilibrium with five millimeters of carbon monoxide is mixed with nitric oxide in solution.
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From page 26... ...
The determination of j4, the velocity constant for the rate of dissociation of the first nitric oxide from hemoglobin, can be carried out by simple classical methods. The reaction is so slow that if a dilute solution of NO hemoglobin is rotated in a tonometer containing carbon monoxide at one atmosphere pressure and rotated for several hours, the time course of reaction and the value of the unimolecular constant can be arrived at from spectroscopic determinations at 3-hourly intervals.
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From page 27... ...
J W.: The determination of the velocity constants of the four successive reactions of carbon monoxide with sheep haemoglobin, Proc.
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From page 28... ...
It is an old creed that these two compounds are physico-chemically similar, but this creed must now go, for the more intimately one studies them, the more frequent are the differences that one finds. Perhaps the most dramatic instance thereof is that the velocity of combination of the first oxygen molecule with hemoglobin is independent of temperature, whereas the velocity constant for the combination of the first carbon monoxide molecule with hemoglobin has a very clearcut dependence on temperature.
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From page 29... ...
I do not know whether or not this is a possible explanation of the phenomena, but I was profoundly struck by the great difference in pH effect on the different velocity constants, particularly the contrast between the 1'~ and the Z,4. This certainly suggests that there is a different kind of coupling with the heme-linked acid groups in these two cases On the other hand, if, as most of us have generally assumed, all of the heme groups are initially equivalent before any reaction has occurred, it is a little difficult for me to see offhand why this should be so.
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From page 30... ...
When any three ligand molecules combine with hemoglobin carbon monoxide, oxygen or nitric oxide—then for some reason a change in the configuration occurs which makes iron atoms behave independently. We do, indeed, now have a very considerable body of evidence which indicates that when either three or four ligand molecules are combined with hemoglobin, the hemoglobin then behaves like myoglobin, the iron atoms all behaving; in the same play and independently of one another This makes it seem unlikely that one out of the four iron atoms was, before combination, different from the others.
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From page 31... ...
This difference is in the velocity constant, i.e., it persists until regeneration is complete.
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From page 32... ...
32 PART I STRUCTURE OF HEMOGLOBIN of course, on the amount of the enzyme—to vary the rate of reduction of oxyhemoglobin to reduced hemoglobin.
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From page 33... ...
In the case of muscle hemoglobin, myoglobin, it may be that the recrystallized condition represents a final conformational state wh ich can only be further changed by denaturation itself. The smaller Bohr effect observable in the metmyog;lobin-fluoride reaction reappears unchanged with the hemoprotein reconstituted from hemin and apomyoglobin.5 The binding of the heme to the globin has usually been discussed from two points of view: either to suggest a mechanism for heme-heme interaction or the Bohr effect, or to account for the remarkable property of ~ This review has been based in part upon research, now in progress, that is supported by grants from the National Science Foundation ( G23 09 )
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From page 34... ...
However, some observations and quantitative data bear on the more general question of the configuration of the heme, quite apart from the chemical nature of the group or groups in the protein to which the iron atom is attached, and quite apart from the role these may play be ~ ~ ~ 1 — — _ _ ~ _ ~ in determining the interaction effects. This kind of evidence will first reviewed under three headings that represent extreme structural types: a)
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From page 35... ...
In this structure the iron in hemoglobin is regarded as forming a classical octahedral complex with four bonds in a plane to the pyrrole N-atoms, a fifth bond to the protein on one side of the porphyrin ring, and a sixth bond on the other side to a coordinated water molecule: id N e.g., Globin Fe—HERO / I At N The magnetic susceptibility data,10 which establish that hemoglobin and methemoglobin can form octahedral complexes, taken in conjunction with the recent theoretical developments in ligand field theory which account for the stability of transition metal compounds in general,~~~-~~3 make the inclusion of a water molecule to complete the octahedral coordination shell an extremely reasonable assumption. This hydrate structure was first discussed in some detail by Hauro~vitz in 1935,14 and, seeking direct experimental confirmation, he later extended Zeynek's observations on the drying of hemoglobin.45~~6~i' He showed that with oxyhemoglobin scarcely any change in the absorption spectrum occurs, and ever at very low pressures (0.1 mm Hg)
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From page 36... ...
If salvation effects were significant, then on the basis of these observations alone no choice could be made between the hydrate structure, a structure in which another unidentified labile group is attached to the iron, and a structure in which the sixth coordination position is unoccupied. At present there is some evidence to suggest that the water does play an essential role beside that of a potential ligand for the iron.
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From page 37... ...
ethyl n-propyl propyl butyl Ferroheme 9.43 9.14 9.04 Horse Myoglobin 5.30 5.04 4.15 3.00 Bovine Hemoglobin 3.9 6 — 3.48 1.70 Human Hemoglobin 4.11 3.52 1.80 The most interesting feature is the sharp drop in the affinity of hemoglobin for the tertiary butyl isocyanide, which is not reflected in the results with ferroheme. This sharp drop was ascribed to steric hindrance.
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From page 38... ...
On the other hand, in its coiled configuration, it would project to the same extent as tertiary butyl isocyanide, but it would appear necessary to postulate an additional complementary hollow to contain the extra methyl group. The difficulties involved in comparing a series of primary, secondary and tertiary alkyl ligands may be illustrated by considering Table III.
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From page 39... ...
Characteristic of this hypothesis that heme-heme interaction has a steric origin is the trend in thermodynamic data to be expected from such a mechanism. If the first ligand molecule to combine reduced the steric hindrance for the second, and so on, it would be predicted that in comparing data for the first and last oxygen molecules the heats of reaction should show a trend to a more favorable value, since more er~ergy would be required to dilate the molecule for the entry of the first; whereas the entropy change should show a trend to a less favorable value, because the entry of the last oxygen molecule would be accompanied by a smaller increase in the freedom of the molecule in the neighborhood of the heme.
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From page 40... ...
The structure shown differs from Conant's original suggestion in having a water molecule coordinated to the iron atom; this removes the objection that hemoglobin does Proximal HN~1 Fe+(H20) G Iobin Distal N~NH FIG.
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From page 41... ...
The general features of reactions involving crevice structures will be considered from two points of view; first, the trend in thermodynamic data to be expected from such a reaction, and secondly, the effect such ~ structure has on the pH dependence of the affinity for a ligand. In considering thermodynamic data a useful comparison with cytochrome c can be made, since a crevice configuration has beers fully established for this hemoprotein.
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From page 42... ...
The reversible heat denaturation of proteins is characterized by unfavorable heat changes and large and favorable entropy changes; for example, Anson and Mirsky found for trypsin that the heat of reversible thermal denaturation was + 68 kcals/mole, and the entropy change +213 e.u.4i From the figures in Table VI, it seems reasonable to conclude that, if the above interpretation is correct, crevice opening plays little or no role in the formation of the ferrimyoglobin complex. No thermodynamic data are available for the corresponding reaction with hemoglobin, but it is known that the corresponding data for the ionizations of ferrihemoglobin and ferrimyogiobin, and for the formation of their fluoride complexes, are very similar indeed.
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From page 43... ...
According to the crevice mechanism Ka would be identified with KAY, and Kb with Ken Kuy; whereas according to the heme-linked ionization mechanism Ka would be identified with Kp, and Kb with Kr A consideration of the relative magnitudes of Kit and Kb shows that it is nevertheless possible in certain circumstances to distinguish between the two mechanisms. For a significant fraction of the hemoprotein to be present in the crevice configuration the equilibrium constant KCr must be greater than unity.
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From page 44... ...
should to a first appro~cimatior~ be independent of the nature of the ligand, whereas according to the heme-linked ionization mechanism Kit (identified with Kp) would be expected to vary from one ligand to another, since the ionizing group H+X is understood to be linked in some way to the iron atom, i.e.
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From page 45... ...
Nevertheless a difficulty arises in the wide variability in the hemoglobin from different species, the variability being manifested not only in the aidinitv for oven, but also in the -- -d ~ ~ D -- magnitude and pH range of the Bohr effect.45~40 To -~ BIAS Air Ai ~ ;~^ .
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From page 46... ...
There is little reason though to believe that the combination of small ligands is subject to any steric hindrance. This structure with one iron-protein bond is the most satisfactory, and although there is no unequivocal experimental evidence that the sixth coordination position is occupied by a water molecule in the absence of an added ligand, theoretical considerations support this as a very reasonable assumption.
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From page 47... ...
J W.: The determination of the velocity constants of the four successive reactions of carbon monoxide with sheep haemoglobin, Proc.
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From page 48... ...
They may be bound in such a fashion that there is ample space for the iron atoms to combine with other ligands. Ir1 a hemoglobin article written a few years ago~ I discussed the problem of the hemaffinic (heme-linked)
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From page 49... ...
DISCUSSION 49 respect. The principal reason for the general belief that imidazole groups are involved, is the high histidine content of mammalian globins.
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From page 50... ...
So far this has given us a picture of the electron density distribution in the haemoglobi~ ' molecule in projection on a plane, at a resolution of about 3 A This picture can be proved to be correct, but on account of the great thickness of the molecule and the consequent overlapping of its components in proj ection we have been unable to recognize in it any polypeptide chains or haem groups.
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From page 51... ...
Note again that bright spots appear only in positions corresponding to bright patches in the diffraction pattern of the single molecule. If the four molecules are replaced by a large number in a regular t~vo-dimensional array, as in the pattern of phthalocyanin illustrated in figure 5, the spots become sharper and form a completely regular
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From page 52... ...
pattern. The distance between the spots is inversely proportional to the distc~nce between the molecules, but their brightness depends only on the brightr~ess of the corresponding region in the diffraction pattern of the single phthalocvanin molecule.
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From page 53... ...
In each case it enables us to~ find the signs of the different patches or spots, simply by observing whether their brightness goes up or down. It will also be noted that the positions of spots of positive or negative amplitude in the diffraction pattern from two or more parallel molecules coincide with those of the corresponding positive or negative patches in the diffraction pattern of the single molecule.
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From page 54... ...
A projection of a structure on a plane, such as figure 6, is calculated from only part of the diffraction pattern of a crystal, namely the x-ray reflexions from lattice planes lying normal to the plane of projection. Overlapping of atoms can be avoided and complete resolution achieved by extending the x-ray analysis to the entire diffraction pattern from the crystal, which may be visualized as a three-dimensional network of spots of varying brightness.
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From page 55... ...
Instead of a single isomorphous pair, it is necessary to have a series of at least three, and if possible more, isomorphous compounds, each having a heavy atom attached to a different site on the protein molecule. By measuring the amount by which the intensity of each diffraction spot changes in each of these isomorphous substitutions, the value of the phase angle can be determined, and the electron density of the molecule can then be calculated in three dimensions.
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From page 56... ...
. The first attempt at solving this projection of haemoglobin by isomorphous substitution with heavy atoms was made by Green, Ingram and Perutz.4 They prepared compounds in which two of the sulphydryl groups of haemoglobin were combined with parachloromercuribenzoate or with silver ions.
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From page 57... ...
Note the difference in scale between this figure and figure 6. The other picture shows the electron density distribution within ~ haemoglobin molecule suspended in saltfree water (fig.
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From page 58... ...
3. Blocked methaemoglobin + 2 moles of Hg(CH3COO-~2 (mercuridiacetate ~ ~ The first and most crucial task in the actual analysis consists in finding the positions of the heavy atoms, for on their correct location depends the subsequent calculation of the signs or phase angles of the x-ray reflexions.
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From page 59... ...
The positions of the mercury atoms bound to the sulphydryl groups show that these groups are spaced 30 A apart in the haemoglobin molecule. The positions of the heavy atoms having been found, the way was now opened for the calculation of signs.
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From page 60... ...
Lipson of the University of Manchester for his kindness in preparing the optical diffraction patterns shown in figures 2, 3 and 4.
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From page 61... ...
Molecular weights were measured by osmometry, light scattering and sedimentation using Archibald's method. Incidentally, the effect of pH 2 and ionic strength 0.05 on sedimentation was checked in two ways.
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From page 62... ...
The second amino acid residue of the faster-moving component is glutamic acid. The slower component has principally leucine in the second position; slight contamination with glutamic acid is ascribed to the difficulty of obtaining the slow component free from the faster in the descending limb of the electrophoresis cell.
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From page 63... ...
As figure 2 shows, they can be anywhere you like, except that they must be related in pairs by the two-fold axis of symmetry. In order to find their position, we made the para-iodo-nitroso-benzene complex of hemoglobin, hoping that the iodines would label the iron atoms and
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From page 64... ...
FIG. 1.—Perspective drawing of the orientation of the heme groups with respect to the crystal axes and the hemoglobin molecule.
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From page 65... ...
DISCUSSION help us to find their positions in an electron density map. Unfortunately, the results obtained so far have been inconclusive.
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