New Findings in Veterinary Microbiology

New Findings in Veterinary Microbiology

Comparative analysis between compound chromatography and affinity chromatography for the purification of Alpha toxin C.perfringense type A

Document Type : Original Article

Authors
Zahra Heshmati 1
Mohsen Fathi Njafi 2
Gholam hossein Ronaghi 1
Mahmood Ebrahimi 1
1 Department of Chemistry, Mashhad Branch, Islamic Azad University, Mashhad, Iran
2 Department of Chemistry, Mashhad Branch, Islamic Azad University, Mashhad, Iran. And Education and Extension Organization (AREEO), Agricultural Research, Razi Vaccine and Serum Research Institute, Mashhad Branch, Mashhad, Iran
10.22034/nfvm.2024.445263.1229
Abstract
Alpha toxin, sometimes referred to as phospholipase C (PLC), is a significant toxin generated by C. perfringens that possesses both deadly and dermonecrotic properties. All strains (A, B, C, D, E) of C. perfringens generate varying quantities of this toxin, which is recognized as a key various  factor in the development of clostridial myonecrosis.Alpha-toxin is known to significantly influence several human and animal disorders, exhibiting deleterious impacts, specifically on the intestinal system. The primary objective of this research was to conduct a comparative analysis of two distinct purification techniques in order to extract alpha-toxin from C. perfringens type A. The first approach involved the alpha-toxin purification through a series of techniques, including ammonium sulfate precipitation, ion-exchange chromatography using DEAE Sephadex at pH values of 7 and 9, and gel filtration chromatography using Sephadex G-100, while the second approach was implemented using the affinity chromatography. The degree of purity of alpha-toxin was assessed at each stage of the purification process by the utilization of SDS-PAGE.The protein content and hemolysis activity were also quantified at each purification step. Based on the obtained findings, examining these two approaches revealed that the first method yielded a percentage of 88, while the second method yielded percentage of 91.7. The specific activity values obtained from the calculations for the first and second methods were 69170 U/mg and 105.71 U/mg, respectively. Our research shows that affinity chromatography can produce a highly pure alpha toxin with a specific activity of 108700 (HU/mg), produced by Clostridium perfringens type A.
Keywords
Alpha-toxin
Clostridium perfringens type A
Enzymatic activity
Affinity chromatography
column chromatogr

Subjects

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New Findings in Veterinary Microbiology
Volume 7, Issue 2
December 2024
Pages 53-62

  • Receive Date 27 February 2024
  • Revise Date 29 March 2024
  • Accept Date 09 June 2024