Myoglobin Molecule Research Articles

The Self-Diffusion Coefficients of Myoglobin and Hemoglobin in Concentrated Solutions

Abstract The self-diffusion coefficients of myoglobin and hemoglobin have been determined with a modification of the Northrup-Anson procedure in which diffusion is made to occur through a Millipore filter paper. In solutions less concentrated than 6 mm (heme) or about 10% (w/v), the self-diffusion coefficient is found independent of protein concentration. At greater protein concentration the logarithm of the self-diffusion coefficient falls linearly with increasing protein concentration. The decrease in self-diffusion coefficient is most simply related to the volume fraction of the solution occupied by protein. The relation between self-diffusion coefficient and hemoglobin concentration remains continuous and linear to the highest attainable concentration, which is the concentration within the erythrocyte. At this concentration the hemoglobin molecules are close-packed and nearly contiguous. The self-diffusion coefficient of hemoglobin in this environment is only 10-fold less than that at infinite dilution. This and other findings indicate that frictional interactions between myoglobin or hemoglobin molecules are minimal. The numerical values of the self-diffusion coefficient serve to test the molecular mechanism proposed for myoglobin (or hemoglobin)-facilitated oxygen diffusion. The facilitated oxygen flux per unit protein concentration is found to be directly proportional to the protein self-diffusion coefficient, as predicted.

Haem hydration and displacement in the pre-denaturational conformational transition of the myoglobin molecule

In accordance with the concept of a restricted accessibility of water molecules to Fe 3+ in metmyoglobin it is established that the observed temperature dependences of spin-lattice proton relaxation rate ( 1 T 1 ) can be explained by the assumption that there are two conformations in the native state of myoglobin, which differ in the relative position of the haem iron from the nearest surface of the molecule with Δx = 1.0–1.6 A ̊ (where x is the distance of the Fe atom from the centre of the idealized spherical myoglobin molecule of radius R). The determination of Δ x is carried out after an adjustment of the distribution parameters P( z) of the diffusion correlation time τ D, solution of the analytical expression for 1 T 1 , and after allowance for the accessibility of Fe 3+ to the water molecules, fixed by the eccentricity α = x/ R for an idealized spherical model of the myoglobin molecule. The data obtained are compared with the evidence (existing in the literature) for a possible conformational equilibrium within the native state of myoglobin-like molecules. It is shown that proton relaxation, as well as other spin-label methods, can be highly sensitive for studying small-scale conformational changes in protein molecules.

Carboxymethylation of Sperm Whale Myoglobin in the Crystalline State

Abstract Crystals of sperm whale ferrimyoglobin were treated with 0.2 m sodium bromoacetate in concentrated ammonium sulfate solution at pH 6.8 for 10 days. The product obtained after dissolving the crystals retained most of the physical properties of the unmodified protein, except for the development of a small number of electrophoretic components representing an increment of about six to eight net negative charges on the average, judged at pH 9.2. The most fully studied preparation contained 6.4, 2.7, 2.0, and 0.8 residue per molecule of histidine, 1,3-dicarboxymethylhistidine, 3-carboxymethylhistidine, and 1-carboxymethylhistidine, respectively. The amino-terminal valyl residue was largely alkylated. Between 1 and 2 residues of lysine were alkylated. No modification of methionine was detected. After removal of heme the modified protein was subjected to cleavage by trypsin and chymotrypsin and the peptides were isolated by chromatography on ion exchange resins and, as required, by paper chromatography or paper electrophoresis. The state of alkylation of 10 of the 12 histidyl residues was established by quantitative estimation of the yields of appropriate tryptic and chymotryptic peptides. The state of residue 12 was determined qualitatively, and that of residue 113 surmised by difference. The following residues were recovered as unmodified histidine: 24, 48, 82, 93, and 97. Residue 119 was recovered primarily in the unmodified form but partly as 1-carboxymethylhistidine. The following residues were recovered primarily or exclusively as 1,3-dicarboxymethylhistidine: 12, 81, 114, and 116. Residue 116 was recovered partly as 3-carboxymethylhistidine. Residue 36 was recovered in high yield exclusively as 3-carboxymethylhistidine. Without exception these results appear to conform to the implications of the crystalline structure reported by Kendrew and Watson when account is taken of the geometry of interactions between neighboring myoglobin molecules in the crystal lattice.

Determination of the orientation and position of the myoglobin molecule in the crystal of seal myoglobin

The orientation of the myoglobin molecule in the crystal of seal myoglobin has been determined, by using the rotation function (Rossmann & Blow, 1962) to compare the 5.8 Å resolution data from sperm whale myoglobin with the 5 Å data from seal myoglobin obtained by Scouloudi (1969). The position of the molecule in the unit cell has been found using the Q-functions defined by Tollin (1966 a). The position of the haem group obtained in this way is in good agreement with that obtained by Scouloudi (1969) from a 5 Å resolution electron density map obtained using the isomorphous replacement map. A three-dimensional electron density map has been obtained by calculating phases for a whale myoglobin molecule correctly located in the seal myoglobin cell and applying these to the observed seal structure amplitudes. This map has been compared with Scouloudi's published results and indicates a good agreement between the two investigations.

X-ray crystallographic studies of seal myoglobin: The molecule at 6 Å and 5 Å resolution

A preliminary three-dimensional study of seal myoglobin ( a = 57.87 A ̊ , b = 29.64 A ̊ , c = 106.38 A ̊ , β = 102 °15′, space group A2, four molecules per unit cell) is described. hkl reflexions to a resolution of 5 Å from crystals of native protein and from two isomorphous derivatives, the aurichloride and the mercuri-iodide, were measured on an automatic three-circle diffractometer. Relative y coordinates of the heavy atoms were determined from a three-dimensional correlation vector synthesis (6Å). From these, and from x and z co-ordinates, previously determined from refinement of the centro-symmetric projection (Scouloudi & Prothero, 1965), “best” phases for the protein were calculated for all reflexions within a radius of 5 Å −1; mean figure of merit < m > = 0.79. Electron density syntheses, calculated first at 6 Å resolution and then including all data within the 5 Å limit, have clearly outlined the course of the polypeptide chain and the position of the haem-group. The results show that the tertiary structure of the seal myoglobin molecule, as previous work had indicated (Scouloudi, 1960), is very similar to that of the sperm whale myoglobin molecule. The synthesis also indicates the positions of some side chains. Difference electron density syntheses (5 Å), between the native protein and each of the two derivatives, were computed using in each case the best phases of the protein. They revealed the positions of the heavy atoms on the seal myoglobin molecule; these are indicated on a balsa wood model of the molecule. Results of this study are discussed in the light of the recently determined amino-acid sequence of seal myoglobin (Bradshaw & Gurd, 1969). A preliminary comparison with sperm whale myoglobin, to the extent that is permitted by the low resolution, has been attempted.

Immunological studies of duck myoglobin.

SummaryPurified duck myoglobin was prepared by ammonium sulfate precipitation and ion-exchange chromatography. Its sedimentation constant was estimated to be approximately 1.75. A specific precipitating antiserum to duck myoglobin was prepared in rabbits. This antiserum did not form precipitates with duck plasma, hemoglobin, liver extract or with components of crude muscle extracts other than myoglobin. No immunologic differences were detectable between duck cardiac or skeletal muscle myoglobin, and none were noted between myoglobins of goose, duck, or chicken present in muscle extracts. Chicken pectoral muscle lacked myoglobin. Precipitin data suggest that three regions on the duck myoglobin molecule act as combining sites or determinants for the rabbit serum. The myoglobin content of several avian muscles was measured immunologically. Skeletal muscle of newly hatched ducklings was deficient in myoglobin content, while cardiac muscle of these immature birds contained almost one-half the adult amount 2 da...

Binding of mercuri-iodide and related ions to crystals of sperm whale metmyoglobin

The binding of murcuri-iodide to crystals of sperm whale metmyoglobin has been investigated by difference Fourier methods. There are two sites of attachment, one in the non-polar interior of the molecule close to, and parallel with, the heme group, and the other in a polar environment between two neighboring myoglobin molecules. In each case the bound group is planar trigonal HgI3−. The insertion of HgI3− at the internal site must necessarily involve considerable shifts of side chains from their normal positions, since the native structure does not contain a cavity large enough to accommodate this group.In parallel investigations it is shown that tri-iodide I3− also binds at the internal site, but mercuri-bromide HgBr3− and iodide I− do not. To summarize these results together with those of other workers, the internal site accepts HgI3−, AuI3−, I3− and Xe, but does not accept HgBr3− or I−.

The nature and configuration of the mercuri-iodide ion in the seal myoglobin derivative

Mercuri-iodide, an ion derived from K 2 HgI 4 , has been used successfully with seal myoglobin, as well as with other crystalline proteins, in preparing isomorphous derivatives, but the nature of the substituent group is not known. The following work was undertaken with the object of determining the structure of the group which becomes attached to the molecule of seal myoglobin. The investigation is confined to the centro-symmetric projection extending to 2 A resolution. Three sets of h 0 l data were employed, measured respectively from crystals of native protein, of the mercuri-iodide and of the gold chloride derivative. The method of isomorphous replacement has been used in a twofold way. (a) From the gold chloride derivative, first refined, preliminary signs were allotted to the structure amplitudes of the protein, and (b) from these protein signs and the observed intensity changes produced by the mercuri-iodide derivative signs could be given to the mercuri-iodide structure factors without any assumptions about the nature of the group. The resulting electron density map favoured a plane trigonal arrangement of one mercury and three iodines projected end-on. Approximate positions of the individual atoms of the group were refined by least squares and were used in a ( F observed — F calculated ) synthesis which revealed the existence of a second site of substitution in the myoglobin molecule. Extensive refinement showed the mean occupancies of the two groups to be 0·54 and 0·17, respectively. The group at the main site appears to be almost planar and is oriented so that its projection is very nearly parallel to the projected plane of the haem group in the myoglobin molecule. The second group, because of the lower occupancy, is less accurately determined but it appears to have a similar configuration.

The myoglobin molecule and its possible structural alterations in diseased states of skeletal and cardiac muscle.

The myoglobin molecule and its possible structural alterations in diseased states of skeletal and cardiac muscle.

The Mechanism of Metmyoglobin Oxidation

From the Division of Physical Chemistry, Commonwealth ScientiJic and Industrial Research Organization, Melbourne, Australia (Received for publication, September 27, 1962) The chemical behavior of metmyoglobin has received con- siderable attention, notably by Keilin and Hartree (1) and by George (2) and George and Irvine (3-5), on the grounds that it has the same prosthetic group as peroxidase and catalase and reacts with II&&, but at a rate more convenient for kinetic investigation. Although the mechanism of HzOz decomposition has proved t.o be different, this in itself is of interest if we can correlate the observed chemical behavior with the nature of the amino acid side chains which surround the iron-porphyrin- histidine-water complex. For such studies the myoglobin molecule is particularly appropriate, since its structure is known with more certainty than that of any other metalloprotein (6). By a combination of electron spin resonance, manometric, and moderately rapid spectrophotometric techniques, applied at low temperatures to slow the reactions, we have found that the complexity of metmyoglobin oxidation is considerably greater than generally believed. The present paper is concerned mainly with the formation and subsequent reaction of the com- plex described by Keilin and Hartree (1) which is characterized by an absorption peak at 545 mp, and with the extent of osida- tion of the protein in the vicinity of the iron atom. In a later paper we shall discuss the nature of the free radical observed by Gibson, Ingram, and Nicholls (7). As an essential part of the argument of the present paper, we show that the free radical is formed extensively at pH 6 and provide evidence for the mech- anism of its appearance and subsequent loss. We wish to point out that the conclusions reached do not necessarily apply below pH 6 or above pH 8.

Electron microscopic investigations of the subunit of haemoglobin and the myoglobin molecule

Haemoglobin and myoglobin have been studied with the help of the negative contrast technique. Uranyl acetate at a pH slightly above 4 has been used as contrasting agent. Under these conditions the haemoglobin molecule dissociates into its four subunits, as is clear from their similarity with the myoglobin molecule. Both the haemoglobin subunit and the myoglobin molecule appear like oval rings, the length of the ring being between 45 and 50 A and its breadth nearly 40 A. The thickness of the ring lies between 25 and 30 A. In some cases the specimens contained more complex patterns than the ones just described. The closed structure of the molecule seemed to have opened up and one explanation of this may be that pH changes occur during the evaporation of the solvent and the buffer salts.

On the structure of sperm whale myoglobin: I. The amino acid composition and terminal groups of the chromatographically purified protein

The hemoprotein that comprises 96% by weight of the protein in crystalline sperm whale myoglobin may be resolved into at least five components by chromatography on the carboxylic resin IRC-50. Four of the five heme-containing components (representing 92% of the protein) possess identical amino acid compositions; the fifth component has not been obtained free of non-heme protein but appears to be very similar to, if not identical with, the composition of the other heme-containing components. The amino acid analyses have shown that the myoglobin molecule contains 153 amino acid residues and possesses a molecular weight of 17,816. Sixty-five per cent of the heme-containing protein is represented by two of the components, designated as components TV and V, and these have been isolated in quantity for structural study. Quantitative methods have been used to demonstrate that components IV and V of sperm whale myoglobin contain a single polypeptide chain with valine at the amino-terminus and glutamine at the carboxyl-terminus.

On the structure of sperm whale myoglobin: III. Amino acid sequences of the smaller tryptic peptides containing aromatic residues

Six of the eleven aromatic amino acid residues in the sperm whale myoglobin molecule are present in relatively small peptides that are constituents of the soluble fraction of tryptic hydrolysates of the protein. Two of these peptides are dipeptides ; their structure was assumed to be defined by the specificity of trypsin. The amino acid sequences of the remaining four peptides, two tripeptides and two hexapeptides, were determined by Edman degradation to be: Phe-Asp-Arg; Leu-Phe-Lys; Ala-Leu-Glu-Leu-Phe-Arg; and Glu-Leu-Gly-Tyr-Gly-Glu-(NH2). The last of these peptides represents the carboxyl-terminal sequence of the protein.

Life span of myoglobin

The myoglobin content of the skeletal muscles of the rat was found to vary between 0.7 and 1.2 mg./g. fresh muscle. The content of cytochrome c which is extractable with water was found to vary between 0.0122 and 0.0187 mg./g. fresh muscle. The corresponding figures for the acid-extractable cytochrome fraction are 0.0141 and 0.0174. Prolonged irradiation with ultraviolet light did not influence the myoglobin and cytochrome c content. Incorporation of C 14 into myoglobin I and II does not significantly differ. The activity of 1 millimole glycine present in the globin of myoglobin of glycine-2-C 14-injected rats is much larger than the corresponding activity of hemin. The pattern of activity decay with time suggests the presence of two populations of myoglobin molecules of different life spans. One has a half-life of 20 days, the other one of 80–90 days. The hemoglobin of our rats had a half-life of 50–60 days. A significant effect of irradiation with 300 rad. of Co 60 γ-rays on C 14 incorporation into the hemin of myoglobin was observed, not, however, on incorporation into the globin moiety.

The crystal structure of myoglobin. VI. Seal myoglobin

Myoglobin from the common seal ( Phoca vitulina ) when crystallized from ammonium sulphate forms monoclinic crystals with space group the unit cell, a = 57·9Å, b = 29·6Å, c = 106·4Å, β = 102°15', contains four molecules. The method of isomorphous replacement has been used in an investigation of the centrosymmetric b -axis projection in which it has been possible to determine signs for nearly all the h0l reflexions having spacings greater than 4Å. Three independent heavy-atom derivatives were employed and the signs so determined have been used to compute a map of the electron density projected on the (010) plane. This projection has been interpreted in terms of the molecule of sperm-whale myoglobin, as deduced by Bodo, Dintzis, Kendrew &amp; Wyckoff (1959) from a three-dimensional Fourier synthesis to 6Å resolution. The results of the interpretation show that the two myoglobin molecules are very similar in form (tertiary structure) in spite of the differences in their amino-acid composition. The relative orientation of the two unit cells with respect to the myoglobin molecule is given and a comparison is made of the positions of the heavy atoms in each molecule.

The crystal structure of myoglobin, V. A low-resolution three-dimensional Fourier synthesis of sperm-whale myoglobin crystals

The study of type A crystals of sperm-whale has now been extended to three dimensions by using the method of isomorphous replacement to determine the phases of all the general X-ray reflexions having d &gt; 6 Å, and a three-dimensional Fourier synthesis of the electron density in the unit cell has been computed. Data were obtained from the same derivatives which had been used in the previous two-dimensional study (Bluhm, Bodo, Dintzis &amp; Kendrew 1958), in the course of which the x and z co-ordinates of the heavy atoms had been determined. Several methods were used to determine the y co-ordinates from the three-dimensional data; with a knowledge of all three co-ordinates of each heavy atom it was possible to establish the phases of nearly all the reflexions by a graphical method. The three-dimensional Fourier synthesis was evaluated on a high-speed computer from these phases and from the observed amplitudes of the reflexions. A resolution of 6 Å was chosen because it should clearly reveal polypeptide chains having a compact configuration such as a helix. The electron-density map was in fact found to contain a large number of dense rod-like features which are considered to be polypeptide chains, probably helically coiled. In addition, a very dense flattened disk is believed to be the haem group with its central iron atom. Finally it was possible to identify the boundaries of the protein molecules by locating the intermolecular regions containing salt solution. An isolated myoglobin molecule has dimensions about 45 x 35 x 25 Å and within it the polypeptide chain is folded in a complex and irregular manner. For the most part the course of the chain can be followed, but there are some doubtful stretches, presumably where the helical configuration breaks down; a crude measurement of the total visible length of chain suggests that about 70% of it may be in a helical or some similarly compact configuration. The haem group is near the surface of the molecule.

The crystal structure of myoglobin IV. A Fourier projection of sperm-whale myoglobin by the method of isomorphous replacement

Two methods were used to attach heavy atoms to specific sites on the myoglobin molecule in type A crystals: (i) the use of specific reagents for the haem group, including iso cyanides and nitroso compounds, (ii) the crystallization of the protein in the presence of various ions, especially mercuri-iodide, aurichloride, mercury diammine, and p -chloro-mercuri-benzene sulphonate. The first method was not completely successful, largely because most of the reagents were so readily displaced by oxygen: the haem group was, however, located by this means. By the second method isomorphous replacements were successfully achieved at four different sites; the chemical mechanism of attachment is in no case well understood. The classical method of isomorphous replacement was used to determine the signs of the hOl (real) reflexions out to spacings of 4 A. x - and z -co-ordinates of the heavy atoms were found by computing difference-Patterson projections, and the signs of the protein reflexions were deduced in the usual manner by relating the signs of the calculated heavy-atom structure factors to the changes of amplitude caused by the introduction of the heavy atom. As a cross-check, signs were independently determined from each of the isomorphous replacements in turn. An electron-density projection of the protein along y was computed; it included all terms of spacing greater than 4 A, but could not be interpreted in terms of chemical structure owing to the degree of overlap inevitable in such a projection (the axis of projection being 31 A). However, the relative positions of the two protein molecules in the unit cell were established by means of a salt-water difference-Fourier projection. To obtain further information about the structure of the molecule it will be necessary to work in three dimensions; it was with this end in view that so large a variety of isomorphous replacements was investigated.

A three-dimensional model of the myoglobin molecule obtained by x-ray analysis.

To understand how a protein performs its individual biological function, it is essential to know its three-dimensional structure. As early as 1934, J.D. Bernal and Dorothy Hodgkin (then Dorothy Crowfoot) showed [Bernal, J. D. & Crowfoot, D. Nature 133, 794–795 (1934)] that proteins, when crystallized, would diffract X-rays to produce a complex pattern of spots. They knew that these patterns contained all the information needed to determine a protein–s structure but, frustratingly, that information could not be deciphered. By comparing patterns from crystals containing different heavy-metal atoms, Max Perutz and colleagues devised the approach that was to solve this riddle. In 1958, J. C. Kendrew et al. applied Perutz–s technique to produce the first three-dimensional images of any protein - myoglobin, the protein used by muscles to store oxygen.