The most important component in red blood cells ishemoglobin, hemoglobin is composed ofGlobinandhemoglobinCombined.The biosynthesis of globin is the same as that of common proteins.Heme is an iron porphyrin compound, a cofactor of hemoglobinmyoglobin、Cytochrome、Peroxidase、catalaseChild base of etc.Heme, which is involved in hemoglobin synthesis, is mainly found in the immature red blood cells andReticulocyteMedium synthesis.[1]
Heme is derived fromacetic acidOr the thin crystalline growth from chloroform pyridine glacial acetic acid is brown in transmitted light and steel blue in reflected light.
Every one in the human bodyhemoglobinIt is composed of four hemes (also called ferrous protoporphyrin) and one globin in the middle. Each heme is composed of four pyrrole subunits, and the center of the ring is aFerrous ion。Each globin has four polypeptide chains, and each polypeptide chain is connected with a heme to form a monomer, or subunit, of hemoglobin.The four subunits of hemoglobin can be automatically assembled into αtwoβtwoForm of.
The four peptide chains that make up globin are different. Adults can be composed of two alpha chains and two beta chains, called HbA.The fetus is composed of two alpha chains and two gamma chains, called HbF.It was replaced by HbA shortly after birth.That is, hemoglobin is composed of one globin and four hemes, and a peptide chain of globin combines with one heme to form a subunit of hemoglobin, and the four subunits are combined with each other by salt bonds between and within subunits.The iron in the middle of each heme group can combine with oxygen to form oxyhemoglobin.The combination or dissociation with oxygen will affect the formation or breaking of salt bonds, which will change the quaternary structure of Hb and its affinity with oxygen, which is the basis of the S-shaped oxygen dissociation curve and the Bohr effect.
Extraction determination
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Extraction principle
When the pH of hemoglobin is lower than 3.0, the combination of heme and globin is the most loose. At this time, organic solvent is addedacetoneTo denature and coagulate globin, dissolve heme in acetone, and add appropriate amount ofTannic acidorSodium acetatePure heme crystals can be obtained, and then washed with ethanol ether to obtain refined heme.Heme has the maximum absorption at wavelength 385, which can be directly determined by colorimetry.[2]
(2) Separate red blood cells and take 10 mL of anticoagulant pig bloodCentrifugal tubeCentrifuge at 3000 r/min for 15 min, pour out the supernatant (plasma), collect red blood cells, and wash them twice with 0.9% NaCl.Washing method: use proper amount of normal salineRed Blood Cells Suspension, stir evenly, centrifuge, collect erythrocyte precipitation, and measure the volume of erythrocyte (mL).
(3) Hemolytic addition ofDeionized water0.25 times 95% ethanol, stirred for 30min, red blood cells swelled with water, and hemoglobin was released.
(4) After separating hemoglobin for hemolysis, add chloroform 0.15 times the volume of red blood cells, stir for 15min, centrifugate, and obtain the sediment containing hemoglobin.
(5) Separation of heme Add 0.15% sodium bisulfite solution equal to the volume of red blood cells into the above sediment to dissolve it, then add acetone 4 times the volume of red blood cells, stir evenly, adjust the pH to 3 with 0.1 mol/L hydrochloric acid solution to fully separate globin and heme (the extraction time is more than 10 min), centrifuge at 4000r/min for 10 min, and discard the precipitation,The supernatant is the heme acetone solution.
(6) The acetone solution of purified heme is concentrated in vacuum and recovered. The concentrate is dissolved in 0.1 mol/L NaOH, centrifuged at 3000 r/min for 10 min, and the supernatant is collected.Adjust the pH to 4-5 with 0.1 mol/L hydrochloric acid, precipitate the heme, collect the precipitate, wash and precipitate with water to neutral, vacuum dry at 50 ℃ to constant weight, and obtain the heme.Determine the weight of heme product (mg).[2]
2. Heme assay
Dissolve the heme standard and the extracted heme product with 0.1 mol/L NaOH, adjust to the appropriate concentration, measure the absorbance at 385nm, and calculate the extraction rate and purity of heme in the sample.[3]
Synthetic decomposition
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biosynthesis
The main component of red blood cells is hemoglobin, accounting for 32% of its wet weight and 97% of its dry weight.Hemoglobin is composed of globin and heme.Heme is not only the cofactor of Hb, but also myoglobinCytochrome、PeroxidaseThe auxiliary heme can be synthesized in a variety of cells in the body, and the heme involved in hemoglobin composition is mainly in the young red blood cells andReticulocyteMedium synthesis.The biosynthesis of globin is the same as that of common proteins.The basic raw materials for heme synthesis in vivo are glycineSuccinyl coenzyme AAnd Fe2+。[4]
Synthesis process of heme
(1) Production of δ - amino - γ - levulinic acid: in mitochondria,glycineAnd succinyl CoA under the catalysis of ALA synthetase to generate ALA.This reaction requires pyridoxal phosphate as a coenzyme,ALA synthase is the rate limiting enzyme for heme synthesis.
(2) Formation of porphyrinogen: after the formation of ALA, it diffuses to the cytoplasm. Under the action of ALA dehydratase, two molecules of ALA dehydrate and condense to form one molecule of porphyrinogen (PBG).
(3) Generation of uroporphyrinogen III and coproporphyrrogen III: under the synergetic catalysis of the cytoplasmic tetramolecular porphyrinogen deaminase and uroporphyrinogen III synthase, the uroporphyrinogen III is synthesized by deamination and condensation, and then through the action of uroporphyrinogen decarboxylase, the uroporphyrinogen III is generated.
(4) Heme production: fecoporphyrinogen III diffuses back into mitochondria.Protoporphyrinogen Ⅸ is generated under the catalysis of coproporphyrnogen oxidationdecarboxylase, and then protoporphyrinogen Ⅸ is generated through the action of oxidase.The latter and FeB produce heme under the catalysis of heme synthase.Heme is transferred from mitochondria to cytoplasm and combines with globin to form hemoglobin.[4]
Metabolic decomposition
containHeme proteinMetabolism in mammals requires:
① The hydrophobic products produced by porphyrin ring shearing were treated;
② Retain and use the iron contained, so that it can be reused.The life cycle of red blood cells is about 120 days. Senile cells are recognized through membrane changes and are recognized by extravascularReticuloendothelial systemdevour.After globin chain denaturation, heme is released into the cytoplasm;Globin is degraded into its amino acids and reused to meet the needs of general metabolism.[5]
Figure 1 (formation of heme to bilirubin)[5]
Figure 1 describes the process of heme metabolism.Heme is mainly degraded by enzymes in the endoplasmic reticulum of reticuloendothelial cells, which requires the participation of oxygen molecules and NADPH.Heme oxygenase(heme oxygenase) has two isomers,Type I is substrate inducible type, and type II is constitutive type.The enzyme catalyzes the shearing of a methylene bridge that links two pyrrole groups containing vinyl substituents.
One methylene carbon is converted to carbon monoxide, which is the only endogenous source of carbon monoxide in the human body.Some carbon monoxide is released through the respiratory tract. Therefore, measuring the exhaled carbon monoxide can be used as an indicator to measure the individual heme degradation.The oxygen in carbon monoxide and the oxygen in the new derivative lactam ring are all derived from oxygen molecules.By stoichiometry, for each ring sheared, the reaction requires 3mol oxygen.Heme oxygenase can only use heme as substrate, and iron may participate in shear reaction.Therefore, free protoporphyrin Ⅸ is not its substrate.Linear tetrapyrrole biliverdin Ⅸ is formed by heme oxygenase.Biliverdin Ⅸ is reduced to bilirubin Ⅸ by biliverdin reductase.[5]
Function
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Combine with oxygen
The process of heme combining with oxygen is a very magical process.First, one oxygen molecule combines with one of the four subunits of heme, and the structure of globin changes after combining with oxygen. This change makes it easier for the second oxygen molecule to find another subunit of heme to combine with the first oxygen molecule, and its combination will further promote the combination of the third oxygen molecule,And so on until the four subunits constituting heme bind to four oxygen molecules respectively.The same is true of the process of oxygen release in tissues. The departure of one oxygen molecule will stimulate the departure of another until all oxygen molecules are completely released. This interesting phenomenon is called synergistic effect.[6]
Due to the synergistic effect of heme molecular structure, the binding curve of heme and oxygen is S-shaped. Within a specific range, with the change of oxygen content in the environment, the binding rate of heme and oxygen molecules has a dramatic change process. The oxygen concentration in the tissues of the organism and the oxygen concentration in the lung tissue are just on both sides of this mutation, so in the lung tissue,Heme can fully combine with oxygen, and can fully release oxygen molecules carried in other parts of the body.However, when the oxygen content in the environment is very high or low, the oxygen binding curve of heme is very gentle, and the huge fluctuation of oxygen concentration can hardly make the binding rate of heme and oxygen change significantly. Therefore, even if healthy people breathe pure oxygen, the ability of blood to carry oxygen will not improve significantly. From this perspective,For healthy people, the psychological implication of oxygen inhalation is far greater than its physiological effect.When the red blood cells in the blood are destroyed too much, the liver load increases, and the transport, binding and excretion of liver cells are obstructed, or the extrahepatic biliary tract is blocked, the concentration of bilirubin in the blood increases and jaundice occurs.
Other functions
In addition to carrying oxygen, heme can also combine with carbon dioxide, carbon monoxide and cyanide ion in exactly the same way as oxygen, except for the firmness of the combination. Once carbon monoxide and cyanide ion combine with heme, it is difficult to leave, which is the principle of gas poisoning and cyanide poisoning,In this case, other substances with stronger ability to combine with these substances can be used for detoxification. For example, carbon monoxide poisoning can be treated by intravenous injection of methylene blue.[7]
As medicine and health care products, it has important biological functions such as carrying oxygen, storing oxygen, promoting oxidation and reduction, and conducting electron transfer.It can be used as raw material for semi synthetic hematoporphyrin (HP) and its derivatives (HPD), protoporphyrin sodium.It can also be used as meat food additive, mainly as colorant.[7]
