Document Type : Original Research Article


1 Research Institute of Applied Science, Academic Center of Education, Culture, and Research (ACECR), Tehran, Iran

2 Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO) Tehran, Iran

3 Research Institute of Applied Science, Academic Center of Education, Culture and Research (ACECR), Tehran, Iran

4 Department of Chemical Engineering, Mahshahr Branch, Islamic Azad University, Mahshahr, Iran



Today, two serious issues threaten human life, such as water pollution with hydrogen sulfide and the emerging corona disease. The special structure of hemoglobin of worms can remove hydrogen sulfide and carry a large amount of oxygen at the same time, and therefore, it is a suitable candidate for solving these two problems. Accordingly, the hemoglobin of Eisenia foetida worms was extracted by homogenization and several centrifuges and filters, and then the dimensions of hemoglobin were measured by dynamic light scattering technique, and the concentration and number of disulfide bonds in hemoglobin were investigated. The results showed that hemoglobin purity after the third stage of centrifugation and the second filtration was 56.2 and 91.3%, the measured diameter was 155.5 and 596.9 nm, the concentration of hemoglobin was 0.015 mol/liter, and the number of disulfide bonds was 120, respectively. According to the results of the examination, the extracted hemoglobin, having a large number of disulfide bonds, can react with hydrogen sulfide, and having very large dimensions, it is suitable for carrying a lot of oxygen.

Graphical Abstract

Extraction of Hemoglobin from Eisenia Foetida Worms


Main Subjects

[1] Shi X, Shao M, Zhang L, Ma Y, Zhang Z. Screening of genes related to sulfide metabolism in Urechis unicinctus (Echiura, Urechidae) using suppression subtractive hybridization and cDNA microarray analysis, Comparative Biochemistry and Physiology Part D: Genomics and Proteomics; 2012 Sep 1; 7(3):254-9.  
[2] Bailly X, Vinogradov S. The sulfide binding function of annelid hemoglobins: relic of an old biosystem?, Journal of inorganic biochemistry; 2005 Jan 1; 99(1):142-50.
[3] Younas F, Mustafa A, Farooqi ZU, Wang X, Younas S, Mohy-Ud-Din W, Ashir Hameed M, Mohsin Abrar M, Maitlo AA, Noreen S, Hussain MM. Current and emerging adsorbent technologies for wastewater treatment: trends, limitations, and environmental implications, Water; 2021 Jan 18; 13(2):215.
[4] Numoto N, Nakagawa T, Kita A, Sasayama Y, Fukumori Y, Miki K. Structure of an extracellular giant hemoglobin of the gutless beard worm Oligobrachia mashikoi, Proceedings of the National Academy of Sciences; 2005 Oct 11; 102(41):14521-6.
[5] Girasole M, Arcovito A, Marconi A, Davoli C, Congiu-Castellano A, Bellelli A, Amiconi G. Control of the active site structure of giant bilayer hemoglobin from the Annelid Eisenia foetida using hierarchic assemblies, Applied Physics Letters; 2005 Dec 5; 87(23). 
[6] Chirita M, Grozescu I, Taubert L, Radulescu H, Princz E. Fe2O3–nanoparticles, physical properties and their photochemical and photoelectrochemical applications, Chemical Bulletin; 2009 Jan; 54(68):1-8.
[7] Shishikura F. The complete amino acid sequences of four globins from the land leech Haemadipsa zeylanica var. japonica, Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology; 2004 Apr 1; 137(4):443-54.  
[8] Zal F, Leize E, Lallier FH, Toulmond A, Van Dorsselaer A, Childress JJ. S-Sulfohemoglobin and disulfide exchange: the mechanisms of sulfide binding by Riftia pachyptila hemoglobins, Proceedings of the National Academy of Sciences; 1998 Jul 21; 95(15):8997-9002.
[9] Román-Morales E, Pietri R, Ramos-Santana B, Vinogradov SN, Lewis-Ballester A, López-Garriga J. Structural determinants for the formation of sulfhemeprotein complexes, Biochemical and biophysical research communications; 2010 Oct 1; 400(4):489-92.
[10] Yuasa HJ, Green BN, Takagi T, Suzuki N, Vinogradov SN, Suzuki T. Electrospray ionization mass spectrometric composition of the 400 kDa hemoglobin from the pogonophoran Oligobrachia mashikoi and the primary structures of three major globin chains, Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology; 1996 Sep 5; 1296(2):235-44. 
[11] Jouan L, Taveau JC, Marco S, Lallier FH, Lamy JN. Occurrence of two architectural types of hexagonal bilayer hemoglobin in annelids: comparison of 3D reconstruction volumes of Arenicola marina and Lumbricus terrestris hemoglobins, Journal of Molecular Biology; 2001 Jan 26; 305(4):757-71.
[12] Meunier C, Andersen AC, Bruneaux M, Le Guen D, Terrier P, Leize-Wagner E, Zal F. Structural characterization of hemoglobins from Monilifera and Frenulata tubeworms (Siboglinids): First discovery of giant hexagonal-bilayer hemoglobin in the former “Pogonophora” group, Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology; 2010 Jan 1; 155(1):41-8. 
[13] Liu S, Dai Z, Chen H, Ju H. Immobilization of hemoglobin on zirconium dioxide nanoparticles for preparation of a novel hydrogen peroxide biosensor, Biosensors and Bioelectronics; 2004 Apr 15; 19(9):963-9.  
[14] Li L, He X, Chen L, Zhang Y. Preparation of core‐shell magnetic molecularly imprinted polymer nanoparticles for recognition of bovine hemoglobin, Chemistry–An Asian Journal; 2009 Feb 2; 4(2):286-93.
[15] Zhao YG, Li H, Xu W, Luo J, Xu RA. An overview of the fibrinolytic enzyme from earthworm, Chinese Journal of Natural Medicines; 2010; 8(4):301-8.   
[16] Zolghadri S, Saboury AA, Amin E, Moosavi-Movahedi AA. A spectroscopic study on the interaction between ferric oxide nanoparticles and human hemoglobin, Journal of the Iranian Chemical Society; 2010 Jul; 7:S145-53.
[17] Moghadam HN, Banaei A, Bozorgian A. Biological Adsorption for Removal of Hydrogen Sulfide from Aqueous Solution by Live Eisenia Foetida Worms, Advanced Journal of Chemistry, Section B: Natural Products and Medical Chemistry; 2022; 4:144-57.  
[18] Ochiai T, Enoki Y. The molecular architecture and the subunits of earthworm (Eisenia foetida) hemoglobin, Comparative Biochemistry and Physiology Part B: Comparative Biochemistry; 1981 Jan 1; 68(2):275-9.
[19] Royer WE, Zhu H, Gorr TA, Flores JF, Knapp JE. Allosteric hemoglobin assembly: diversity and similarity, Journal of Biological Chemistry; 2005 Jul 29; 280(30):27477-80.
[20] Elmer J, Palmer AF, Cabrales P. Oxygen delivery during extreme anemia with ultra-pure earthworm hemoglobin, Life sciences; 2012 Oct 29; 91(17-18):852-9.
[21] Strand K, Knapp JE, Bhyravbhatla B, Royer Jr WE. Crystal structure of the hemoglobin dodecamer from Lumbricus erythrocruorin: allosteric core of giant annelid respiratory complexes, Journal of molecular biology; 2004 Nov 12; 344(1):119-34.
[22] Wajcman H, Kiger L, Marden MC. Structure and function evolution in the superfamily of globins, Comptes rendus biologies; 2009 Feb 1; 332(2-3):273-82.
[23] Petersen MT, Jonson PH, Petersen SB. Amino acid neighbours and detailed conformational analysis of cysteines in proteins, Protein engineering; 1999 Jul 1; 12(7):535-48.
[24] Shishikura F, Snow JW, Gotoh T, Vinogradov SN, Walz DA. Amino acid sequence of the monomer subunit of the extracellular hemoglobin of Lumbricus terrestris, Journal of Biological Chemistry; 1987 Mar 5; 262(7):3123-31.
[25] Kraus DW, Wittenberg JB. Hemoglobins of the Lucina pectinata/bacteria symbiosis. I. Molecular properties, kinetics and equilibria of reactions with ligands, Journal of Biological Chemistry; 1990 Sep 25; 265(27):16043-53.
[26] Bozorgian A. Investigation of Hydrate Formation Kinetics and Mechanism of Effect of Inhibitors on it, a Review, Journal of Chemical Reviews; 2021 Jan; 3(1): 50-65.
[27] Goffredi SK, Childress JJ, Desaulniers NT, Lallier FH. Sulfide acquisition by the vent worm Riftia pachyptila appears to be via uptake of HS−, rather than H2S, Journal of Experimental Biology; 1997 Oct 1; 200(20):2609-16.
[28] Meiers CW. An Examination of Eisenia fetida Coelomic Fluid for Antimycobacterial Activity, Minnesota State University, Mankato; 2015.
[29] Girguis PR, Childress JJ, Freytag JK, Klose K, Stuber R. Effects of metabolite uptake on proton-equivalent elimination by two species of deep-sea vestimentiferan tubeworm, Riftia pachyptila and Lamellibrachia cf luymesi: proton elimination is a necessary adaptation to sulfide-oxidizing chemoautotrophic symbionts, Journal of experimental biology; 2002 Oct 1; 205(19):3055-66.
[30] Rajesh A, Zimmerman D, Spivack K, Abdulmalik O, Elmer J. Glutaraldehyde cross‐linking increases the stability of Lumbricus terrestris erythrocruorin, Biotechnology progress; 2018 Mar; 34(2):521-8.
[31] Meyers MB, Fossing H, Powell EN. Microdistribution of interstitial meiofauna, oxygen and sulfide gradients, and the tubes of macro-infauna, Marine Ecology Progress Series; 1987 Feb 5; 223-41.
[32] M. Meyers, E. Powell, H. Fossing, Movement of oxybiotic and thiobiotic meiofauna in response to changes in pore-water oxygen and sulfide gradients around macro-infaunal tubes, Marine Biology 98 (1988) 395-414. Meyers MB, Powell EN, Fossing H. Movement of oxybiotic and thiobiotic meiofauna in response to changes in pore-water oxygen and sulfide gradients around macro-infaunal tubes, Marine Biology; 1988 Jun; 98:395-414.  
[33] Vismann B. Physiology of sulfide detoxification in the isopod, Saduria (Mesidotea) entomon; 1991.
[34] Rizzi M, Wittenberg JB, Coda A, Fasano M, Ascenzi P, Bolognesi M. Structure of the sulfide-reactive hemoglobin from the clam Lucina pectinata: Crystallographic analysis at 1.5 Å resolution, Journal of molecular biology; 1994 Nov 17; 244(1):86-99.
[35] Yeh E, Pinsky BA, Banaei N, Baron EJ. Hair sheep blood, citrated or defibrinated, fulfills all requirements of blood agar for diagnostic microbiology laboratory tests, PloS one; 2009 Jul 3; 4(7):e6141.
[36] Zimmerman D, DiIusto M, Dienes J, Abdulmalik O, Elmer JJ. Direct comparison of oligochaete erythrocruorins as potential blood substitutes, Bioengineering & Translational Medicine; 2017 Jun; 2(2):212-21.
[37] Abele-Oeschger D, Oeschger R. Hypoxia-induced autoxidation of haemoglobin in the benthic invertebrates Arenicola marina (Polychaeta) and Astarte borealis (Bivalvia) and the possible effects of sulphide, Journal of Experimental Marine Biology and Ecology; 1995 Apr 18; 187(1):63-80.
[38] Nourani S, Ghourchian H, Boutorabi SM. Magnetic nanoparticle-based immunosensor for electrochemical detection of hepatitis B surface antigen, Analytical biochemistry; 2013 Oct 1; 441(1):1-7.
[39] Ghorbannezhad H, Moghimi H, Taheri RA. Enhanced biodegradation of phenol by magnetically immobilized Trichosporon cutaneum, Annals of Microbiology; 2018 Aug; 68:485-91.
[40] Savla C, Munoz C, Hickey R, Belicak M, Gilbert C, Cabrales P, Palmer AF. Purification of lumbricus terrestris mega-hemoglobin for diverse oxygen therapeutic applications, ACS biomaterials science & engineering; 2020 Aug 12; 6(9):4957-68.
[41] Wang X, Gai Z, Yu B, Feng J, Xu C, Yuan Y, Lin Z, Xu P. Degradation of carbazole by microbial cells immobilized in magnetic gellan gum gel beads, Applied and Environmental Microbiology; 2007 Oct 15; 73(20):6421-8.
[42] Swain S, Behera A, Beg S, N Patra C, C Dinda S, Sruti J, EB Rao M. Modified alginate beads for mucoadhesive drug delivery system: an updated review of patents, Recent Patents on Drug Delivery & Formulation; 2012 Dec 1; 6(3):259-77.
[43] Chinthapally K, Blagg BS, Ashfeld BL. Syntheses of Symmetrical and Unsymmetrical Lysobisphosphatidic Acid Derivatives, The Journal of Organic Chemistry; 2022 Jul 27; 87(15):10523-30.