Rapid purification of porcine colostral whey lactoferrin by affinity chromatography on single-stranded DNA-agarose. Characterization, amino acid composition and N-terminal amino acid sequence.
We have determined that the major iron-binding and DNA-binding protein in porcine colostral whey is lactoferrin. This lactoferrin was purified to homogeneity in one chromatographic step using immobilized single-stranded DNA-agarose. Although different in chromatographic behavior from human lactoferrin, the porcine lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, size-exclusion chromatography (TSK-4000SW), and reverse-phase (phenyl) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels under reducing conditions showed the purified lactoferrin to be a single protein (silver-stained) of 78 kDa. Apolactoferrin purified in this manner bound iron and displayed a UV/VIS absorption spectrum indistinguishable from that of human lactoferrin. The molar absorption coefficient of hololactoferrin was 3.86 x 10(3) M-1 at 465 nm and 1.08 x 10(5) M-1 at 280 nm. Affinity elution analyses of the purified lactoferrin on immobilized DNA revealed that the affinity of this protein for DNA was independent of bound iron. Porcine lactoferrin was recognized by antibodies directed against human lactoferrin and bovine lactoferrin. The amino acid composition and N-terminal amino acid sequence analysis (30 residues) revealed a high degree of sequence homology with human, equine and bovine lactoferrin. These results demonstrate the effectiveness of immobilized DNA as a rapid and simple lactoferrin purification procedure and demonstrate the presence of a lactoferrin in porcine colostral whey with a high degree of sequence homology to human lactoferrin.
Hutchens
T W
TW
USDA/ARS Children's Nutrition Research Center, Department of Pediatries, Baylor College of Medicine, Houston 77030.
Magnuson
J S
JS
Yip
T T
TT
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D003126:Colostrum / Q000032:analysis*; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007781:Lactoferrin / Q000235:genetics / Q000302:isolation & purification*; D007782:Lactoglobulins / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D000596:Amino Acids; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D007782:Lactoglobulins; D007781:Lactoferrin