Direct AFM force mapping of surface nanoscale organization and protein adsorption on an aluminum substrate.

We investigate the nanoscale organization of a superficially hydroxylated Al substrate and its effect on subsequent protein adsorption using atomic force microscopy (AFM). For this purpose we used a mode which allows a direct mapping of a variety of surface properties (adhesion, elasticity, dissipation, etc.) to be probed simultaneously with topographical images. The hydroxylation treatment leads to a drastic modification of the surface morphology, owing to the formation of AlOOH compounds. In air, AFM images revealed the formation of regular nanorod-like structures randomly distributed, inducing the appearance of nanoporous domains on the surface. In buffer solution, prior to the adsorption of proteins, the surface nanoscale organization is preserved, mainly due to the chemical stability of AlOOH compounds under these conditions. The adsorption of proteins on the obtained nanostructured surface was performed using either a globular (ß-lactoglobulin) or a fibrillar (collagen) protein and by modulating the adsorbed amount through the incubation time or the concentration of proteins in solution. At low amounts, collagen adsorbs on the whole surface without preferential localization. The surface topography remains similar to the bare surface, while significant changes were evidenced on adhesion and elasticity maps. This is due to the fact that the surface became adhesive and less stiff, owing to the presence of a soft and hydrated protein layer. By contrast, ß-lactoglobulin tends to diffuse into the nanoporous domains, leading to their filling up, and the surface is blurred with a thick and dense protein layer upon increasing the amount of adsorbed molecules. Our findings demonstrate the interest in using AFM for surface mapping to investigate the mechanism of protein adsorption at the nanoscale on materials with high surface roughness.

Size-selective separations of biological macromolecules on mesocylinder silica arrays.

In order to control the design functionality of mesocylinder filters for molecular sieving of proteins, we fabricated tight mesocylinder silica nanotube (NT) arrays as promising filter candidates for size-exclusion separation of high-concentration macromolecules, such as insulin (INS), α-amylase (AMY), ß-lactoglobulin (ß-LG), and myosin (MYO) proteins. In this study, hexagonal mesocylinder structures were fabricated successfully inside anodic alumina membrane (AAM) nanochannels using a variety of cationic and nonionic surfactants as templates. The systematic design of the nanofilters was based on densely patterned polar silane coupling agents ("linkers") onto the AAM nanochannels, leading to the fabrication of mesocylinder silica arrays with vertical alignment and open surfaces of top-bottom ends inside AAM. Further surface coating of silica NTs hybrid AAM with hydrophobic agents facilitated the production of extremely robust constructed sequences of membranes without the formation of air gaps among NT arrays. The fabricated membranes with impermeable coated layers, robust surfaces, and uniformly multidirectional cylinder pores in nanoscale sizes rapidly separate large quantities of proteins within seconds. Meanwhile, comprehensive factors that affect the performance of the molecular transport, diffusivity, and filtration rate through nanofilter membranes were discussed. The mesocylinder filters of macromolecules show promise for the efficient separation and molecular transport of large molecular weight and size as well as concentrations of proteins.

Separation of lactoferrin-a and -b from bovine colostrum.

Bovine lactoferrin was separated into lactoferrin-a and lactoferrin-b from bovine colostrum. Lactoferrin-a was eluted at 0.38 M NaCl and lactoferrin-b was eluted at 0.43 M NaCl by carboxymethyl cation-exchange chromatography at pH 7.7, 0.05 M phosphate buffer. The molecular weights were estimated at 84,000 for lactoferrin-a and 80,000 for lactoferrin-b. Lactoferrin-a contents were 258.0 mg/L and lactoferrin-b contents were 524.3 mg/L of colostrum for cow 19. From colostrum to normal milk, total lactoferrin was from 17.1 to 129.4 mg/L during the normal lactational period; however, lactoferrin did not separate clearly into lactoferrin-a and lactoferrin-b. The lactoferrin-a measured from six cows was 258.0, 114.0, 112.8, 64.0, 59.7, and 22.4 mg/ L and the lactoferrin-b 524.3, 331.8, 184.7, 170.7, 129.3, and 44.0 mg/L, respectively. The average was 105.2 mg (31.3%) for lactoferrin-a and 230.8 mg (68.7%) for lactoferrin-b.

Interaction of human lactoferrin with DNA: one-step purification by affinity chromatography on single-stranded DNA-agarose.

Human lactoferrin has been purified to apparent homogeneity with high recovery (greater than 95%) in one chromatographic step using immobilized single-stranded DNA-agarose. Human colostral whey, at neutral pH, was loaded onto columns of DNA-agarose; a single purified protein, lactoferrin, was eluted using elevated ionic strength buffers. The lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, and reverse-phase (C18) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels and silver staining showed the purified lactoferrin preparation to contain a single protein of 78 kD with intact immunologic determinants. Similar results were obtained before and after iron saturation of the colostral whey proteins. Apolactoferrin purified in this manner was shown to bind iron with high affinity. These results demonstrate the effectiveness of immobilized DNA as one of the most rapid and complete lactoferrin purification procedures described thus far.

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.

One-step isolation of lactoferrin using immobilized monoclonal antibodies.

Immunoaffinity columns made with monoclonal antibodies to either human or bovine lactoferrins were prepared to isolate human lactoferrin or bovine lactoferrin from milks by a single chromatographic step. Recoveries of human lactoferrin and bovine lactoferrin were 98 and 97%, respectively. The human lactoferrin recovered from defatted human colostrum was 98% pure with 93% iron-binding capacity. Amount of recovered bovine lactoferrin, as well as purity and iron-binding capacity, varied widely depending on the source of bovine milks and pretreatments (particularly pasteurization temperature). The best source to isolate bovine lactoferrin was raw skim milk yielding a protein 97% pure and with a 99% iron-binding capacity. Thus, immunoaffinity chromatography provides an effective one-pass isolation of highly pure human or bovine lactoferrin with reasonable recovery and iron-binding capacity.

Preliminary crystallographic studies on bovine lactoferrin.

The purification of bovine lactoferrin, its crystallization at low ionic strength, and preliminary X-ray crystallographic data are reported. The crystals, which grow from a two-phase system, are radiation-stable and suitable for a medium-resolution X-ray analysis. They are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 138.4 A, b = 87.1 A, c = 73.6 A, and one protein molecule in the asymmetric unit.

Isolation and characterisation of lactoferrin separated from human whey by adsorption chromatography using Cibacron Blue F3G-A linked affinity adsorbent.

A new one-step procedure for isolating lactoferrin from human whey using Cibacron Blue affinity chromatography is described. Characteristics of the isolated lactoferrin include pI 5.5-6.1 and differential migration on SDS-polyacrylamide gel electrophoresis of apo and iron saturated lactoferrin. The concentration of lactoferrin in breast milk varies with time after parturition but the iron saturation remains relatively constant with time.

Isolation of lactoferrin and its concentration in sows' colostrum and milk during a 21-day lactation.

Levels of lactoferrin, an Fe-binding protein with bacteriostatic properties, were determined in the colostrum and milk of Yorkshire sows during a 21-d lactation. Lactoferrin levels averaged 1,100 to 1,300 micrograms/ml near the time of farrowing, then declined sharply during the first week of lactation. Concentration of lactoferrin showed considerable variation among sows, but not among teat positions (anterior to posterior). A method for isolating high purity swine lactoferrin is described.

Studies on human lactoferrin by electron paramagnetic resonance, fluorescence, and resonance Raman spectroscopy.

Investigations of metal-substituted human lactoferrins by fluorescence, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy confirm the close similarity between lactoferrin and serum transferrin. As in the case of Fe(III)- and Cu(II)-transferrin, a significant quenching of apolactoferrin's intrinsic fluorescence is caused by the interaction of Fe(III), Cu(II), Cr(III), Mn(III), and Co(III) with specific metal binding sites. Laser excitation of these same metal-lactoferrins produces resonance Raman spectral features at ca. 1605, 1505, 1275, and 1175 cm-1. These bands are characteristic of tyrosinate coordination to the metal ions as has been observed previously for serum transferins and permit the principal absorption band (lambda max between 400 and 465 nm) in each of the metal-lactoferrins to be assigned to charge transfer between the metal ion and tyrosinate ligands. Furthermore, as in serum transferrin the two metal binding sites in lactoferrin can be distinguished by EPR spectroscopy, particularly with the Cr(III)-substituted protein. Only one of the two sites in lactoferrin allows displacement of Cr(III) by Fe(III). Lactoferrin is known to differ from serum transferrin in its enhanced affinity for iron. This is supported by kinetic studies which show that the rate of uptake of Fe(III) from Fe(III)--citrate is 10 times faster for apolactoferrin than for apotransferrin. Furthermore, the more pronounced conformational change which occurs upon metal binding to lactoferrin is corroborated by the production of additional EPR-detectable Cu(II) binding sites in Mn(III)-lactoferrin. The lower pH required for iron removal from lactoferrin causes some permanent change in the protein as judged by altered rates of Fe(III) uptake and altered EPR spectra in the presence of Cu(II). Thus, the common method of producing apolactoferrin by extensive dialysis against citric acid (pH 2) appears to have an adverse effect on the protein.