Binding characteristics of folate to high affinity folate binding protein purified from porcine serum.
High affinity folate binding protein (HFBP) in porcine serum was purified 2,000-fold to a specific activity of 1.4 nmol of tetrahydrofolic acid (THF) bound per mg of protein, using folic acid (FA) coupled EAH-Sepharose gel affinity chromatography. Binding activity of purified HFBP to folate was examined by ultrafiltration method linked to high-performance liquid chromatography with electrochemical detection or to liquid scintillation counting. Binding affinity of HFBP to folate was characterized by dissociation constants (Kd): 13, 17, and 31 pM for tritiated FA (3HFA), THF, and N5-methyltetrahydrofolic acid (5MF), respectively. FA, THF, and 5MF significantly inhibited binding of HFBP to 3HFA, and according to the magnitude of intensity of the binding inhibition, the order of affinity of each folate was confirmed to be FA > THF > 5MF. Binding activity was rather high and stable for THF and 5MF at pH ranging from 6.0 to 10.0. The binding activity, however, rapidly decreased at pH below 6.0 and over 10.0. No binding activity was observed pH below 3.0 and over 12. Gel filtration analysis showed that the prepared HFBP solution had specific binding activity at around 77 kDa of apparent molecular weight, which was 82 kDa by SDS-PAGE. It is considered that this specific and stable binding enables THF to distribute in porcine plasma.
Natsuhori
M
M
Laboratory of Veterinary Radiology and Radiation Biology, Kitasato University School of Veterinary Medicine and Animal Sciences, Towada, Aomori, Japan.
Okada
M
M
Ida
R
R
Sasaki
K
K
Shimoda
M
M
Kokue
E
E
D000818:Animals; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011956:Receptors, Cell Surface; D013552:Swine
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D005492:Folic Acid