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Biomedical subjects

M F Perutz

Publications and source records attributed to M F Perutz.

At least 55 records · Page 3Linked to original sources
PubMed1990

Frequency of abnormal human haemoglobins caused by C----T transitions in CpG dinucleotides.

A large part of human genetic disease apparently arises from deamination of cytosines in methylated CpG dinucleotides. Their mutation rate is known to be high when C is present as 5-methylcytosine, but is believed to be normal when it is unmethylated. The beta-globin gene contains five, the gamma-globin gene two, and each of the alpha-globin genes contain 35 CpGs. The CpGs in the beta- and gamma-globin genes are methylated, while those in the alpha-globin genes are undermethylated. One would therefore have expected the CpGs to be a frequent source of mutations in the beta- and gamma-globin genes, but not in the alpha-globin genes. In fact, the evidence points to CpGs being a frequent source of mutations in both the alpha- and beta-globin genes. This suggests either that the mutation rates of both methylated and unmethylated CpGs are abnormally high, which conflicts with published evidence, or that there is a finite chance of some CpGs in the alpha-globin genes of certain individuals being methylated and therefore subject to mutation.

Base Sequence↗
PubMed1990

Structure and function of hemoglobin variants at an internal hydrophobic site: consequences of mutations at the beta 27 (B9) position.

We have studied the structure-function relationships in newly discovered hemoglobin (Hb) mutants with substitutions occurring at the tight and highly hydrophobic cluster between the B and G helices in the beta chains, namely, Hb Knossos or beta A27S and Hb Grange-Blanche or beta A27V. The beta A27S mutant has a 50% decrease in oxygen affinity relative to native human Hb A, while the beta A27V mutant has an increased oxygen affinity. We have also engineered the artificial beta A27T mutation through site-directed mutagenesis. This new mutant exhibits functional properties similar to those of Hb A. None of these mutants is unstable. X-ray analyses show that the substitution of Val for Ala may reduce the relative stability of the T structure of the molecule through packing effects in the beta chains; for the beta A27S mutant a new hydrogen bond between serine and the carbonyl O at beta 23 (B5) Val is observed and is likely to increase the relative stability of the T structure in the mutant hemoglobin. However, no significant changes in the crystals were observed for these mutants between the quaternary R and T structures relative to native Hb A. We conclude that small tertiary structural changes in the tight hydrophobic B-G helix interface are sufficient to induce functional abnormalities resulting in either low or high intrinsic oxygen affinities.

Binding Sites↗
PubMed1990

Frequency of abnormal human haemoglobins caused by C----T transitions in CpG dinucleotides.

A large part of human genetic disease apparently arises from deamination of cytosine residues in methylated CpG dinucleotides. Their mutation rate is known to be high when C is present as 5-methyl-cytosine, but is believed to be normal when it is unmethylated. The beta-globin gene contains five, the gamma-globin gene two, and each of the alpha-globin genes contains 35 CpG dinucleotides. The CpG dinucleotides in the beta and gamma-globin genes are methylated, while those in the alpha-globin genes are under-methylated. One would therefore have expected the CpG dinucleotides to be a frequent source of mutations in the beta and gamma-globin genes, but not in the alpha-globin genes. In fact, the evidence points to CpG dinucleotides being a frequent source of mutations in both the alpha and beta-globin genes. This suggests either that the mutation rates of both methylated and unmethylated CpG dinucleotides are abnormally high, which conflicts with published evidence, or that there is a finite chance of some of these in the alpha-globin genes of certain individuals being methylated and therefore subject to mutation.

Base Sequence↗
PubMed1990

New effectors of human hemoglobin: structure and function.

We describe the actions of two new allosteric effectors of hemoglobin, 2-[4-(3,5-dichlorophenylureido)phenoxy]-2-methylpropionic acid (L35) and 2-[4-(3,4,5-trichlorophenylureido)phenoxy]-2-methylpropionic acid (L345). Each of them binds to two pairs of symmetry-related sites in the central cavity of human deoxyhemoglobin. One pair of sites overlaps with that occupied by bezafibrate [Perutz et al. (1986) J. Am. Chem. Soc. 108, 1064-1078]. The other sites are new, and the pair occupied by L35 is different from that occupied by L345. All the sites are at least 20 A from the site where organic phosphates are bound. L345 is by far the most potent allosteric effector of hemoglobin ever described. At a concentration of 0.1 mM, it raises the P50 of a suspension of red cells by 50%; at 0.2 mM it raises the P50 2.5-fold. At acid pH, it reduces Hill's coefficient to near unity and prevents complete oxygen saturation even under 1 atm of pure oxygen. In azidemethemoglobin at pH 6, it induces a transition to higher spin. These properties are reminiscent of those of teleost fish hemoglobins that exhibit a Root effect. The influence of L35 and L345 and that of organic phosphates on the oxygen affinity are additive, but they compete with chloride. L35 acts more weakly than L345, but can be made to induce the same effects as L345 alone by adding inositol hexaphosphate. Both compounds increase the alkaline and acid Bohr effects. They alter the bimolecular kinetics of CO recombination after a flash by increasing the slowly reacting fraction of hemoglobin in the T state at the expense of the fast-reacting fraction in the R state.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1990

Hemoglobin Warsaw (Phe beta 42(CD1)----Val), an unstable variant with decreased oxygen affinity. Characterization of its synthesis, functional properties, and structure.

In Hb Warsaw Val replaces the Phe normally present at the heme contact position beta 42 (CD1). This variant is unstable, and it readily undergoes methemoglobin formation. In DEAE-cellulose chromatography, the variant hemoglobin co-eluted with Hb A; a partially heme-depleted fraction of the variant, representing 5-6% of the total hemoglobin, eluted separately and in pure form. The heme replete form of Hb Warsaw exhibited decreased oxygen affinity with a normal Bohr effect and normal cooperativity and interaction with 2,3-diphosphoglycerate (DPG). The heme-depleted Hb Warsaw had a higher oxygen affinity than that of Hb A, decreased cooperativity and 2,3-DPG interaction, and a very low alkaline Bohr effect. Gel filtration of the heme-depleted form showed it to exist entirely as alpha beta dimers. Globin chain synthesis by Hb Warsaw-containing reticulocytes followed a balanced alpha/beta ratio. In short-term synthesis experiments, a major portion of incorporated radiolabeled L-leucine was recovered from the dimeric, heme-depleted Hb Warsaw fraction, suggesting that subunit association precedes the incorporation of heme into the beta subunits in the post-synthetic assembly of this hemoglobin. Structural analysis of deoxyhemoglobin containing roughly equal proportions of normal and variant beta chains showed that the replacement leaves a cavity next to the heme that is large enough to hold a water molecule, which may account for the instability of Hb Warsaw. The heme and the pyrrol nearest to ValCD1 tilt into the cavity. The resulting increase in the tilt of the proximal histidine relative to the heme plane, coupled with a possible stretching of the Fe-N epsilon bond may account for the low oxygen affinity.

2,3-Diphosphoglycerate↗
PubMed1990

Frequency of abnormal human haemoglobins caused by C----T transitions in CpG dinucleotides.

A large part of human genetic disease apparently arises from deamination of cytosines in methylated CpG dinucleotides. Their mutation rate is known to be high when C is present as 5-methyl-cytosine, but is believed to be normal when it is unmethylated. The beta-globin gene contains five, the gamma-globin gene two, and each of the alpha-globin genes contain 35 CpG's. The CpG's in the beta-and gamma-globin genes are methylated, while those in the alpha-globin genes are undermethylated. One would therefore have expected the CpG's to be a frequent source of mutations in the beta- and gamma-globin genes, but not in the alpha-globin genes. In fact, the evidence points to CpG's being a frequent source of mutations in both the alpha- and beta-globin genes. This suggests either that the mutation rates of both methylated and unmethylated CpG's are abnormally high, which conflicts with published evidence, or that there is a finite chance of some CpG's in the alpha-globin genes of certain individuals being methylated and therefore subject to mutation.

5-Methylcytosine↗
PubMed1989

Structure and function of human hemoglobin covalently labeled with periodate-oxidized adenosine triphosphate.

Periodate-oxidized adenosine triphosphate (o-ATP), a ribose ring-opened dialdehyde derivative of ATP, reacts specifically with human deoxyhemoglobin to give a single major covalently modified product after reduction with sodium borohydride. This product, designated di-ATP Hb, was isolated using ion-exchange chromatography and shown to have incorporated two molecules of o-ATP/tetramer. Peptide mapping and x-ray crystallography at 2.8-A resolution indicate that a covalent adduct is formed between the ligand and residues Lys-82 EF6 of each beta chain in the organic phosphate-binding site of the molecule. di-ATP Hb exhibits a significantly decreased oxygen affinity (P50 = 20.8 mm Hg versus 5.8 mm Hg control; 50 mM 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol, pH 7.4, 0.1 M C, 20 degrees C). The subunit cooper-activity of di-ATP Hb is also reduced (nmax = 1.9 versus 2.7 control).

Adenosine Triphosphate↗
PubMed1988

Aromatic rings act as hydrogen bond acceptors.

Simple energy calculations show that there is a significant interaction between a hydrogen bond donor (like the greater than NH group) and the centre of a benzene ring, which acts as a hydrogen bond acceptor. This interaction, which is about half as strong as a normal hydrogen bond, contributes approximately 3 kcal/mol (1 cal = 4.184 J) of stabilizing enthalpy and is expected to play a significant role in molecular associations. It is of interest that the aromatic hydrogen bond arises from small partial charges centred on the ring carbon and hydrogen atoms: there is no need to consider delocalized electrons. Although some energy calculations have included such partial charges, their role in forming such a strong interaction was not appreciated until after aromatic hydrogen bonds had been observed in protein-drug complexes.

Benzene↗
PubMed1988

Indirect allosteric effects of a neutral mutation. Structure of deoxyhaemoglobin north Chicago (ProC2(36)beta----Ser).

Haemoglobin North Chicago (ProC2(36 beta----Ser) has an abnormally high oxygen affinity. A survey of other abnormal human haemoglobins with high oxygen affinity has indicated that mutations leading to a cavity in the quaternary T-structure, or to the rupture of any bond in that structure, have raised oxygen affinities, because such mutations loosen the constraints of the T-structure. They do not usually affect the oxygen affinity of the R-structure. Haemoglobin North Chicago aroused our interest because the side-chain of serine is smaller than that of proline by only one carbon atom, and it was hard to conceive how such a small gap should raise the oxygen affinity significantly. Our X-ray study shows that the mutation produces unexpectedly large indirect changes in the T-structure.

Crystallization↗
PubMed1988

LR16, a compound with potent effects on the oxygen affinity of hemoglobin, on blood cholesterol, and on low density lipoprotein.

2-[4-(3,4-Dichlorophenylureido)phenoxy]-2-methylpropionic acid, LR16, combines with two symmetrically related sites in the central cavity of deoxyhemoglobin, 20 A away from the binding site of 2,3-bisphosphoglycerate, and acts as an allosteric effector synergistic with 2,3-bisphosphoglycerate. LR16 (1 mM) raises P50, the partial pressure of oxygen needed to achieve half-saturation with oxygen of a hemolysate of human hemoglobin, about 50 times more strongly than 1 mM 2,3-bisphosphoglycerate. Oral administration of LR16 (at small doses that produced no ill effects) to rats that were fed a diet rich in cholesterol caused substantial reductions of total serum cholesterol and low density lipoprotein-cholesterol, while high density lipoprotein-cholesterol remained unchanged.

Animals↗
PubMed1987

Is there a Root effect in Xenopus hemoglobin?

The reaction of Xenopus hemoglobin with oxygen and carbon monoxide has been reinvestigated over the pH range 8.5-6.0, in the absence and presence of organic phosphates (2,3-diphosphoglycerate or inositol hexakisphosphate), to establish if the tetramer can be stabilized in a T-quaternary state by protons and polyphosphate; the equilibrium and kinetic data indicate that Xenopus hemoglobin does exhibit a Root effect. These new results are discussed with reference to those reported by Bridges et al. [(1985) Resp. Physiol. 61, 125-136] on Xenopus blood and, more generally, to the molecular definition and the structural basis of the Root effect as an extreme form of the Bohr effect.

2,3-Diphosphoglycerate↗
PubMed1987

Influence of anions and protons on the Adair coefficients of haemoglobins A and Cowtown (His HC3(146) beta----Leu).

We have measured the contribution of the alkaline Bohr effect of the C-terminal histidine residues of the beta-chains of haemoglobin A by comparing haemoglobin A with haemoglobin Cowtown in which those histidine residues are replaced by leucine. Oxygenation of a stripped 2.5 mM (haem) solution of haemoglobin A yielded 0.19 H+/haem, while oxygenation of a similar solution of haemoglobin Cowtown produced no change of pH. Oxygen equilibria measured at 60 microM-haem in 0.1 M-Hepes buffer gave an alkaline Bohr effect of -0.21 H+/haem for haemoglobin A and only -0.01 H+/haem for haemoglobin Cowtown, even though its Hill's coefficient was greater than 2 throughout the pH range studied. These results prove that the chloride-independent part of the alkaline Bohr effect is due to the C-terminal histidine residues of the beta-chains. Oxygen equilibria measured in 0.095 M-bis-Tris buffers with minimal chloride or with 0.1 M-chloride showed the contribution of those histidine residues to the alkaline Bohr effect to be about 0.2 H+/haem, independent of chloride concentration. Determination of the individual Adair coefficients in the three different buffers indicated that pH and chloride tend to have their greatest effects at the second or third steps of oxygenation when the change of quaternary structure is most likely to occur; between pH 7 and 9, the fourth Adair coefficient is only very slightly affected by pH and not significantly by chloride.

Anions↗