An easy two-step purification method for human leucocyte myeloperoxidase

Bahram SARKARATI, (Hacettepe Üniversitesi, Tıp Fakültesi, Tıbbi Biyokimya Anabilim Dalı, Ankara, Türkiye)
Tülay AKYOL KARAAĞAÇ, (Hacettepe Üniversitesi, Hastanesi, Kan Transfüzyon Merkezi, Ankara, Türkiye)
Kamer KILINÇ (TOBB Ekonomi ve Teknoloji Üniversitesi, Tıp Fakültesi, Ankara, Türkiye)
Türk Biyokimya Dergisi
2 0
Yıl: 2015 Cilt: 40 Sayı: 5 Sayfa Aralığı: 410 - 416 Metin Dili: Türkçe İndeks Tarihi: 29-07-2022

An easy two-step purification method for human leucocyte myeloperoxidase

Öz:
Amaç: Bu çalışmanın amacı, tek vericiden temin edilen lökositlerden miyeloperoksidaz (MPO) enzimini saflaştırmak için basit, hızlı ve ekonomik bir yöntem geliştirmektir. Çalışmada, insan lökosit miyeloperoksidaz enziminin konkanavalin A-Sefaroz 4B afinite kromatografisi ile CM-Sefadeks katyon değiştirici kromatografisinden oluşan iki basamakta saflaştırılması sunulmuştur.Metod: Saflaştırma çalışmasında, lökoferez ile tek kişiden alınmış insan lökositleri kullanıldı. Lökosit miyeloperoksidazı, %1 oranında HETAB (hekzadesiltrimetilamonyum bromide) içeren fosfat tampon içinde çözünürleştirildi ve santrifügasyonla diğer zarsal fraksiyonlardan ayrıştırıldı. Çözünürleştirilen enzim konkanavalin A-Sefaroz afinite jeline uygulandı ve bağlanan enzim bu jelden metil-a-D-manno-piranozid ile indirildi. Kromatografide MPO aktivitesi içeren fraksiyonlar birleştirildi, diyaliz edildikten sonra CM-sefadeks katyon değiştirici kolona uygulandı. Enzim bu kolondan hafif bazik pH'daki fosfat tampon içinde lineer NaCl gradient ile indirildi.Bulgular: Uygulanan iki kromatografik yöntemle MPO enzimi insan lökositlerinden %70 verimle saflaştırıldı. Enzimin saflığı, Reinheit Zahl (RZ) değeri (A430/A280) ölçülerek control edildi. Reinheit Zahl değeri saflaştırılan MPO için 0.86 olarak hesaplandı. Bu RZ değeri, daha önce literatürde saf enzim için bildirilen değerler (0.82-0.88 arasında) ve saf ticari MPO için belirlenmiş değer (0.84) ile karşılaştırıldığında, bu saflaştırma çalışması ile elde edilen enzimin saflığını gösterir.Sonuç: Önceki saflaştırma metotlarıyla karşılaştırıldığında bu metot daha yüksek geri kazanım oranı ve saflık oranını birlikte sunmaktadır. Lökofrez kaynaklı lökosit kullanımı ve amoniyum sulfat basamağının çıkarılması maliyetin azalması ve zamanın kısalmasını sağlamıştır.
Anahtar Kelime:

Konular: Biyokimya ve Moleküler Biyoloji

İnsan lökosit miyeloperoksidazı saflaştırılması için kolay 2 basamaklı yöntem

Öz:
Objective: The object of this study is to describe a simple, rapid and cost effective method for purification of human leucocyte myeloperoxidase from a single donor. Myelopeoxidase (MPO) was purified by a two step procedure consisting of concanavalin-A Sepharose 4B affinity chromatography followed by CM-Sephadex cation exchange chromatography.Methods: Leucocytes from a single donor collected by leucopheresis were used in purification studies. MPO was solubilized and extracted from leucocytes by homogenization in phosphate buffer containing 1% HETAB (hexadecyltrimethylammonium bromide). MPO containing soluble material was applied onto concanavalin-A Sepharose 4B affinity gel, and was eluted with methyl-aD-manno-piranoside. Fractions with MPO activity were pooled, dialyzed and applied onto CM-sephadex cation exchange gel, and was eluted from the column at weak cationic pH with linear NaCl gradient.Results: By the use of two chromatographic procedures, MPO was purified from human leucocytes with 70% yield. Purity of MPO was checked by determining the Reinheit Zahl (RZ) value (A430/A280). The RZ value of 0.86 indicated that the purified enzyme was highly homogenous as compared to reported experimental values (ranging from 0.82 to 0.88) and pure commercial enzyme with the RZ value of 0.84.Conclusion: In comparison with earlier purification methods, the purification method reported here has higher recovery rate and high purity together. Use of leucocytes with leucopheresis origin help us to omit the leucocyte isolation step and omitting of ammonium sulphate precipitation steps also help us to reduce the cost and is shortened the time of purification.
Anahtar Kelime:

Konular: Biyokimya ve Moleküler Biyoloji
Belge Türü: Makale Makale Türü: Araştırma Makalesi Erişim Türü: Erişime Açık
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APA SARKARATI B, AKYOL KARAAĞAÇ T, KILINÇ K (2015). An easy two-step purification method for human leucocyte myeloperoxidase. , 410 - 416.
Chicago SARKARATI Bahram,AKYOL KARAAĞAÇ Tülay,KILINÇ Kamer An easy two-step purification method for human leucocyte myeloperoxidase. (2015): 410 - 416.
MLA SARKARATI Bahram,AKYOL KARAAĞAÇ Tülay,KILINÇ Kamer An easy two-step purification method for human leucocyte myeloperoxidase. , 2015, ss.410 - 416.
AMA SARKARATI B,AKYOL KARAAĞAÇ T,KILINÇ K An easy two-step purification method for human leucocyte myeloperoxidase. . 2015; 410 - 416.
Vancouver SARKARATI B,AKYOL KARAAĞAÇ T,KILINÇ K An easy two-step purification method for human leucocyte myeloperoxidase. . 2015; 410 - 416.
IEEE SARKARATI B,AKYOL KARAAĞAÇ T,KILINÇ K "An easy two-step purification method for human leucocyte myeloperoxidase." , ss.410 - 416, 2015.
ISNAD SARKARATI, Bahram vd. "An easy two-step purification method for human leucocyte myeloperoxidase". (2015), 410-416.
APA SARKARATI B, AKYOL KARAAĞAÇ T, KILINÇ K (2015). An easy two-step purification method for human leucocyte myeloperoxidase. Türk Biyokimya Dergisi, 40(5), 410 - 416.
Chicago SARKARATI Bahram,AKYOL KARAAĞAÇ Tülay,KILINÇ Kamer An easy two-step purification method for human leucocyte myeloperoxidase. Türk Biyokimya Dergisi 40, no.5 (2015): 410 - 416.
MLA SARKARATI Bahram,AKYOL KARAAĞAÇ Tülay,KILINÇ Kamer An easy two-step purification method for human leucocyte myeloperoxidase. Türk Biyokimya Dergisi, vol.40, no.5, 2015, ss.410 - 416.
AMA SARKARATI B,AKYOL KARAAĞAÇ T,KILINÇ K An easy two-step purification method for human leucocyte myeloperoxidase. Türk Biyokimya Dergisi. 2015; 40(5): 410 - 416.
Vancouver SARKARATI B,AKYOL KARAAĞAÇ T,KILINÇ K An easy two-step purification method for human leucocyte myeloperoxidase. Türk Biyokimya Dergisi. 2015; 40(5): 410 - 416.
IEEE SARKARATI B,AKYOL KARAAĞAÇ T,KILINÇ K "An easy two-step purification method for human leucocyte myeloperoxidase." Türk Biyokimya Dergisi, 40, ss.410 - 416, 2015.
ISNAD SARKARATI, Bahram vd. "An easy two-step purification method for human leucocyte myeloperoxidase". Türk Biyokimya Dergisi 40/5 (2015), 410-416.
Sayfa Oluşturulma Tarihi
22-04-2024 23:58

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