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Effect of Amino Acid Chelate Iron on Heme Synthesis
Abstract In this study, the effect of amino acid chelated iron on the activity of ALA synthetase was studied by in vitro enzymatic assay. The experimental results show that: 1 When the heme concentration in the enzyme reaction system is 510-5 mol/L, it inhibits ALA synthetase. 2 Different iron sources promote the activity of ALA synthetase, and different iron sources have different effects on ALA synthetase activity. Glycine and ferrous sulfate mixture group> Ferrous sulfate group> Lysine and ferrous sulfate mixture group > Lysine chelated iron group> Glycine chelated iron group. Keywords hemoglobin, ferrous sulfate, lysine chelated iron, glycine chelated iron, synthesis studies on heme metabolism indicate that ALA (δ-amino-γ-levulinic acid, δ-Aminolaevulinic acid) synthetase is The rate-limiting enzyme in the heme synthase system (Gu Tianjue 1988). Studies have shown that heme has feedback inhibition on this enzyme. In general, after the synthesis of hemoglobin, hemoglobin is rapidly combined with globin to form hemoglobin without excessive hemoglobin accumulation, and feedback inhibition by ALA synthase is not caused. If heme synthesis rate is greater than the synthesis rate of globin, excess heme can oxidize to hemin. Studies have shown that the synthesis of ALA synthase can be inhibited when the concentration of heme is 10-6 mol/L, and the activity of ALA synthase can be inhibited when it is 10-5 to -4 mol/L (the physiological normal value of heme is 10-7~-6mol/L), while metharubin is a strong inhibitor of ALA synthetase. Since the ALA synthetase is regulated by the heme content (concentration of the end product), according to the biochemical feedback inhibition theory, a hypothesis is proposed: if there is an amino acid chelate iron in the cell, the structure of the chelate is similar to that of hemoglobin As a competitive analogue, it may bind to the control center of ALA synthetase and compete with the regulatory site, and this binding can only be "similar", this similarity does not affect the change of enzyme activity, thus making ALA synthesis Enzymes are still active in the presence of "more" heme. Based on the above theory and the results of the previous experiments, this experiment was designed to study the effect of amino acid chelated iron on the activity of ALA synthetase through in vitro enzymatic tests, and the effect of amino acid chelated iron on heme synthesis was inferred. Hypotheses provide arguments. 1 Test reagents and instruments ATP, pyridoxal phosphate, reduced glutathione, ALA standards, and heme were purchased from Sigma. Succinyl-CoA synthetase (10 U/mg at 25[deg.] C.) was purchased from Boehringer Mannheim, Germany. Sucrose, EDTA, Tris, glycine, succinic acid, MgCl2, trichloroacetic acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, p-dimethylaminobenzaldehyde, glacial acetic acid, perchloric acid, mercury dichloride, concentrated hydrochloric acid, Ethyl acetoacetate, glacial acetic acid, n-butanol, and anhydrous sodium sulfate are all domestically produced, and the product grade is pure analytical reagent. Glycine chelated iron and lysine chelated iron as laboratory preparations. Glass homogenizer, HZS-H constant-temperature water bath oscillation box, Japan Hitachi SCR20BC 20000RPM refrigerated centrifuge, Japan Shimadzu UV-2401PC ultraviolet spectrophotometer. 2 Technical route of the experiment 2.1 Preparation of mitochondria separation Homogenate: Weigh 85.55g of sucrose, 0.01g of EDTA, 6.05g of Tris, dissolve in about 800ml of distilled water, add 4.25ml of 0.1mol/L HCl, and finally make up to 1000ml with distilled water. , Store in a refrigerator at 0°C. Specific operation: Take fresh porcine liver, wash with pre-cooled (0°C) homogenate medium, dry the surface water with filter paper, and weigh. The liver was cut with scissors, and 8 ml of homogenate medium was added per gram of liver tissue, added to a glass homogenizer, and homogenized in an ice bath. After the liver was crushed, it was removed to a centrifuge tube, centrifuged at 4409 g on a refrigerated centrifuge for 15 min, and the supernatant was aspirated. The supernatant was centrifuged at 13 675 g for 30 min on a refrigerated centrifuge. The supernatant was discarded and the remainder was the mitochondrial fraction. Wash four times with homogenate medium and freeze. When it is used in the future, the mitochondria will be disrupted by several times of alternating lyophilization as a crude enzyme solution. 2.2 The main method for determining the reaction conditions is Wider (1971). Take 2ml of the reaction solution in a test tube. 2ml reaction solution is composed of 100μmol glycine, 100μmol succinate, 10μmol ATP, 3.5μmol CoA, 10μmol MgCl2, 0.25μmol pyridoxal phosphate, 10μmol reduced glutathione. 50 μmol Tris-HCl buffer (pH = 7.2), 1 mg succinyl-CoA synthetase, exact amount of crude enzyme solution. After incubation at 37°C for 30 minutes, 0.5 ml of 25% trichloroacetic acid was added and the reaction was terminated. Sampling analysis ALA content, if ALA synthesis, indicating that the above reaction conditions apply, otherwise it is modified. 2.3 Determination of Factors Influencing ALA Synthetase (Experimental Design) Based on 2.2, according to the following plan, substances that may affect the activity of ALA synthetase were added, and then the production of ALA was measured to study the change of ALA synthase activity. Glycine chelated iron, lysine chelated iron, glycine and ferrous sulfate mixture, lysine and ferrous sulfate mixture, ferrous sulfate, the same concentration above; heme, respectively high and low two Concentration. Based on the physiological normal value of heme and the concentration of ALA synthase inhibitory activity, this test set a high heme concentration of 510-5 mol/L, a low heme concentration of 510-7 mol/L, and glycine chelated iron and Lai. The iron chelated by the amino acid, the mixture of glycine and ferrous sulfate, the mixture of lysine and ferrous sulfate, and the concentration of ferrous sulfate were both set to 510 to 5 mol/L (calculated as iron). 2.4 Determination of ALA Production The principle of the assay method used is: ALA and ethyl acetoacetate are reacted at pH 6.8 and 100°C to form pyrrole derivatives, which react with p-dimethylaminobenzoic acid to form red compounds. , Extraction with chloroform, colorimetric quantification. 2.5 Representation of enzyme activity The amount of ALA produced per unit of time is an ALA synthase activity unit (U). 2.6 Data Processing and Analysis The data obtained from the test were analyzed for variance and multiple comparisons using the GLM procedure in SAS (version 6.12) software. Graphic production using Microsoft Excel. 3 Results and analysis The test results are shown in Table 1 and Figure 1. Different iron sources have different effects on ALA synthase activity. There was no significant difference between the ferrous sulfate group and the mixture of glycine and ferrous sulfate (p>0.05), which was significantly higher than that of other treatment groups (p<0.05), but significantly higher than that of the hyperhemoglobin group (p<0.05). From the measured values, the low heme group was significantly higher than the high heme group. 4 Discussion Feedback inhibition refers to the inhibition of the activity of key enzymes in the reaction system by the final product in the reaction system, and it is one of the important ways to rapidly adjust enzyme activity in living organisms. The result of a series of metabolic reactions in the organism gradually leads to the accumulation of the final product, so that the reaction rate is slowed down or even stopped. When the final product is consumed or transferred to lower the concentration, an environment conducive to the reaction is gradually formed. This constant adjustment is like a constant temperature control. This enzyme is called an allosteric enzyme and the final product is called an allosteric inhibitor. Some researchers used ALA synthetase in the partially purified mammalian liver and found that when the heme concentration was 510-5 mol/L (the physiological normal value of heme was 10-7~10-6 mol/L. ), can directly inhibit the activity of ALA synthetase, visible, heme feedback inhibition of ALA synthase (Ouyang Pei, 1989). Studies on feedback inhibition mechanisms have shown that the inducer is added to the enzyme reaction system, and the inducer binds to the control center of the allosteric enzyme. This combination does not affect the activity of the enzyme, but also prevents the binding of the final product to the allosteric enzyme. This increases the activity of the enzyme. Then, the structure of amino acid chelated iron is very similar to that of hemoglobin. Adding amino acid chelated iron in the heme synthesis reaction can play the role of inducer? Although the activity of ALA synthase measured in the enzymatic reaction system with a heme concentration of 510-5 mol/L (heme) was higher than that of heme (510-7 mol/L) (low heme group) The measured value was lower by 13.32%, but statistically, the difference between the measured values ​​of the hyperhemoglobin group and the low heme group was not significant, which was mainly due to the low statistical sample size (n=2). The results of this experiment indicate that heme has inhibitory effects on ALA synthetase in the heme group, which is consistent with previous studies (Ouyang Pei, 1989). After the addition of a mixture of glycine and ferrous sulfate or ferrous sulfate (concentration 510-5 mol/L) to the reaction system, the measured activity of ALA synthase was significantly higher than that of the low heme group, respectively. 66.01% and 57.70% higher than the low heme group, indicating that the addition of a mixture of glycine and ferrous sulfate or ferrous sulfate in the reaction system promotes ALA synthetase. Although the addition of other iron sources (lysine and ferrous sulfate mixture, glycine chelated iron and lysine chelated iron) to the reaction system, the difference between the activity of ALA synthetase and that of the low heme group was not significant. , But from the specific measurements, the addition of several iron sources increased the activity of ALA synthetase to varying degrees (lysine and ferrous sulfate mixture group, 10.54%; lysine chelated iron group, 6.39%; glycine Chelated iron group, 3.65%). Wider (1971) research using soybean cells showed that the addition of Fe2+ to the enzyme reaction system enhances the activity of ALA synthetase. Vogel's (1960) study of blood taken from iron-deficient ducks showed that the addition of Fe2+ to an in vitro enzyme reaction system enhanced the ability of iron-deficient cells to synthesize heme. At the same time, he also found that Fe2+ and glycine were simultaneously added to the reaction system. The promotion of heme synthesis is stronger than adding glycine or iron alone. The results obtained in this experiment are basically consistent with their results. It is not difficult to find the difference between each enzyme reaction system after adding various iron sources. As can be seen from 2.2, the enzyme reaction system contains glycine (as a raw material to participate in the reaction). After adding ferrous sulfate, the entire reaction system is similar to the mixture of glycine and ferrous sulfate. The pH value of the enzyme reaction system is 7.2. This pH value is very favorable for the formation of amino acid chelated iron. It can be presumed that the complex cationic glycine chelated iron is formed in the enzyme reaction system. The chemical structure of iron chelated by glycine is very similar to that of pyrrole in the porphyrin ring of heme. Glycine chelated iron as a competitive analog may bind to the control center of ALA synthetase, compete for binding to regulatory sites, and thus have an impact on the activity of ALA synthase. The lysine chelated iron has some side differences and the chemical structure is slightly different from the pyrrole on the porphyrin ring in the heme. This effect may not occur, and the results obtained in this experiment also support this point. Since the stability constants of iron chelated by lysine chelated iron and glycine are similar, after the addition of a mixture of lysine and ferrous sulfate to the enzyme reaction system, both types of chelated iron may be formed, but it can be seen from the test results that lysine is present. The effect of coexistence of acid chelated iron and glycine chelated iron is not as great as that of glycine chelated iron alone. This is due to the fact that when Fe2+, lysine, and glycine coexist, more complex compounds are formed or other reasons remain to be determined by further experiments. From the test results, it can be seen that the addition of lysine chelated iron and glycine chelated iron (internal complex salt type) to the reaction system also has an effect on the activity of ALA synthetase, but is not as strong as the first three iron sources, mainly due to the The solubility of amino acid chelated iron (inner salt type) in a solution with a pH of 7.2 (reaction system of this experiment) is low, which is not inconsistent with the results obtained in Chapters 1 and 2 because it is in the small intestine fluid. The solubility of amino acid chelated iron has been greatly improved (Liao Yiping, 1999). The results obtained in this experiment can only be the establishment of the previous hypothesis (if there is an amino acid iron chelate in the cell, the structure of the chelate is similar to that of hemoglobin. As a competitive analogue, it may be combined with the control center of ALA synthetase. , and this combination can only be "similar", this similarity does not affect the changes in enzyme activity, so that ALA synthetase can still provide activity in the presence of "more" heme, providing a strong evidence, To prove the correctness of the hypothesis, more in-depth biochemical research must be conducted. 5 Conclusions (1) When the heme concentration in the enzyme reaction system is 510-5 mol/L, it inhibits ALA synthetase. (2) Different iron sources promote the activity of ALA synthetase, and different iron sources have different effects on ALA synthetase activity. Glycine and ferrous sulfate mixture group> Ferrous sulfate group> Lysine and ferrous sulfate Mixture group> lysine chelated iron group> glycine chelated iron group.