Purification of human plasma fibronectin using immobilized gelatin and Arg affinity chromatography

This protocol describes a method for purification of fibronectin (Fn) from human plasma based on a combination of gel filtration and affinity chromatography steps. Clarified plasma is first loaded onto a Sepharose CL-4B column and unbound material is sequentially purified on columns containing covalently coupled gelatin and Arg. The elution conditions are optimized to obtain a homogeneous preparation of Fn on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the Fn yield is expected to be lower than that obtained using other methods, affinity adsorbents based on gelatin and Arg and gentle elution steps offer advantages including a high purity of the preparation and a correctly folded protein. The preparation can be useful for interaction studies and analysis of biological and immunological activities of Fn.     

Purification of angiotensin-converting enzyme from rabbit lung and human plasma by affinity chromatography

Lisinopril (N alpha-[(S)-1-carboxy-3-phenylpropyl]L-lysyl-L-proline), a potent angiotensin-converting enzyme inhibitor, is an exceptionally selective affinity chromatography ligand for this enzyme. Affinity chromatography furnishes electrophoretically homogeneous enzyme directly from crude homogenates of rabbit lung tissue, a 1,000-fold purification; also, it affords a 100,000-fold enrichment of the more rare human plasma enzyme in a single step. The affinity of angiotensin-converting enzyme for the Sepharose-spacer-lisinopril matrix (Ki matrix = 1 X 10(-5) M) is weak compared to its affinity for free lisinopril (Ki = 1 X 10(-10) M). The capacity of the affinity column is described quantitatively as a function of Ki matrix, lisinopril, and enzyme concentrations. The recovery of bound enzyme is low in chromatography of crude tissue samples (10-40%), although it approaches a reversible process (70-100%) with pure enzyme. The holoenzyme is converted to Zn2+-free apoenzyme to effect removal of lisinopril. In this process, the rate constant for spontaneous dissociation of Zn2+ from free enzyme is 1 X 10(-2) s-1 (t 1/2 = 1 min), which places a lower limit of 3 X 10(-10) M on the dissociation constant of Zn2+ at neutral pH from angiotensin-converting enzyme. The exceptional selectivity of lisinopril as an affinity chromatography ligand for angiotensin-converting enzyme suggests it is among the most specific inhibitors designed for any enzyme.     

Purification of canine plasma renin by affinity chromatography.

An affinity column for the purfication of canine plasma renin was prepared using goat anti-renin (dog kidney) gammaG gloublins. The antiserum was prepared against a purified kidney renin preparation. The anti-renin globulins were coupled to cyanogen bromide activated Sepharose. Using the anti-renin globulin-coupled Sepharose as an immuno-adsorbant, a method was devised allowing purification of plasma renin to a 1,000-fold purity.     

Polylactosamine glycosylation on human fetal placental fibronectin weakens the binding affinity of fibronectin to gelatin

Gelatin-binding chymotryptic fragments from placental fibronectin contain polylactosamine carbohydrates (Zhu, B.C.R., Fisher, S.R., Pande, H., Calaycay, J. Shively, J.E., and Laine, R.A. (1984) J. Biol. Chem. 259, 3962-3970). We have separated polylactosamine-containing gelatin-binding fragments of placental fibronectin from their counterparts containing smaller "complex" N-linked saccharides using Sephadex G-200 gel permeation chromatography. The peptide portions of both fragments have similar amino acid composition and N-terminal sequence (see reference above). The strength of binding of these two glycosylation types of chymotryptic fragments to gelatin differs as shown by the following experiments. 1) Upon urea gradient elution of affinity-bound fibronectin fragments from gelatin-Sepharose chromatography, the apex of the elution peak for polylactosamine-containing fragments occurs at 2.0 M urea while the peak for complex N-linked carbohydrate-containing fragments maximized at 2.5 M urea indicating a tighter binding. Removal of polylactosamine sequences from the former glycopeptide by endo-beta-galactosidase digestion caused the elution peak for this fraction to change from 2.0 to 2.5 M, the same as for the complex N-linked carbohydrate-containing glycopeptide. 2) Competitive displacement experiments give an apparent dissociation constant of polylactosamine-containing fragments at 3 X 10(-9) M whereas this constant for complex carbohydrate-containing fragments is 1 X 10(-9) M. These results indicate that the binding of placental fibronectin to gelatin is weakened by the presence of high molecular weight polylactosamine carbohydrate. To our knowledge this is the first report that the type and extent of glycosylation of a glycoprotein can affect its binding affinity to a proteinacious ligand. Thus, fetal placental fibronectin may have different biological properties than fibronectins containing only the smaller N-linked complex carbohydrate.     

Purification of human immunoglobulin M by affinity chromatography on protamine-sepharose

Human IgM has been isolated from plasma by a simple procedure in high yield. The first step was adsorption to protamine-Sepharose and elution by increasing the ionic strength with NaCl. This was followed by two gel filtration steps resulting in a 98% pure IgM in about 30% yield. A somewhat modified procedure could also be used for purification of IgM from Cohn fraction II + III. The purified IgM was found to have a sedimentation constant in agreement with reported values. In immunoelectrophoresis and isoelectric focusing, purified IgM showed the same behaviour as IgM in plasma. Different fragments of IgM were tested for binding to the protamine-Sepharose adsorbent. IgM_S and Fab were not bound unlike (Fc)₅, indicating that the sites responsible for binding are located in the Fc part and that several Fc parts are necessary for sufficiently strong binding for adsorption.     

Purification of human liver glycoprotein sialyltransferase by affinity chromatography

A simple procedure has been developed for purifying solubilized human liver glycoprotein sialyltransferase (EC 2.4.99.1) 16 000-fold in 4–5% yield. The procedure involves two centrifugation steps, affinity chromatography of the ultrasupernatant fluid on cytidine diphosphate-hexanolamine-agarose followed by gel filtration on Sephadex G-150. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the purified sialyltransferase preparation contains approximately equivalent amounts of three protein bands (with apparent molecular weights of 61 000, 63 000 and 70 000) and is highly purified if not homogeneous.     

Purification of fibronectin using gelatin derivatives

This work compares two distinct methods, column and batch, for the purification of fibronectin, from different plasma fractions, using gelatin and one of its derivates (Hemoce, Behring). The yields of both techniques at quantitative as well as qualitative levels (levels of immunologically active fibronectin), are evaluated. The data indicate that better conservation of immunological immunological characteristics is obtained with the use of gelatin derivatives (Hemoce). The plasma fraction does not have significant influence on the process yields.     

Fluid-phase interaction between human plasma fibronectin and gelatin determined by fluorescence polarization assay

Previous studies of the binding properties of fibronectin (Fn) have utilized methods whereby one or the other macromolecule was immobilized on a solid phase. In order to examine the interaction between human plasma Fn and gelatin in solution, the latter was labeled with fluorescein isothiocyanate (FITC) whose fluorescence polarization (P) served as a sensitive indicator of the formation of soluble complexes. Changes in P were detectable at Fn concentrations below 10(-9) M and continued up to concentrations above 10(-6) M at pH 7.3 and 25 degrees C. Fractionation of FITC-gelatin by exclusion chromatography and titration of selected fractions revealed a trend towards higher affinity with increasing size. A high-molecular-weight fraction comprised of beta and gamma components and a low-molecular-weight fraction comprised primarily of alpha chains exhibited sigmoidal increases in P (apparent positive cooperativity) with 50% saturation near 10(-9) and 10(-8) M Fn, respectively. By contrast, a 42-kDa chymotrypsin-generated Fn fragment which retains the ability to adhere to gelatin-Sepharose exhibited normal (noncooperative) binding to both gelatin fractions with Kd = 7 X 10(-7) M. In all cases, the increase in P could be reversed by addition of excess unlabeled gelatin or urea. The interaction of FN with FITC-gelatin provides the basis for a fast and sensitive determination of Fn levels in plasma and other fluids. Interference caused by other proteins such as albumin, which has an affinity for the fluorescein moiety, could be minimized by addition of 1.0 M NaCl which had no effect on the interaction between Fn and gelatin.     

Purification of ficin by affinity chromatography

The sulfhydryl proteinase ficin (EC 3.4.4.12) was purified by chromatography on an agarose-mercurial column. Two separate protein fractions were eluted, ficin and mercurificin, both exhibiting enzymatic activity upon activation by excess thiol.     

Purification of antibodies by affinity chromatography

This review focusses on affinity purification of immunoglobulins, a methodology which is a powerful tool to obtain pure and intact antibodies. Affinity techniques allow antibody purification both in a single step chromatographic procedure as well as in complex purification protocols depending on the intention to use the target antibody. The purification strategies for antibodies by interaction with affinity ligands such as antibodies and Fe receptors or low molecular weight compounds are described.     

Purification of alternanase by affinity chromatography

The enzyme alternanase, produced by Bacillus sp. NRRL B-21195, hydrolyzes alternan, a polysaccharide produced by certain strains of Leuconostoc mesenteroides that consists of glucose linked by alternating α(1→6), α(1→3) linkages. The main product of enzymatic hydrolysis by alternanase is a novel cyclic tetrasaccharide of glucose that also has alternating linkages between the glucose moieties. An improved purification scheme for alternanase has been developed that incorporates the use of isomaltosyl units linked to agarose for selectively binding the alternanase enzyme. Bound enzyme was eluted with 0.5 M sodium chloride and was nearly pure after this procedure. When followed by preparative isoelectric focusing, a single band of 117 kDa was measured when the purified protein was analyzed by HPLC size-exclusion chromatography/multiangle light scattering. The purification procedure can be scaled to permit large quantities of enzyme to be purified in high (36%) yield. Electronic Publication     

Purification of human serum angiotensin I-converting enzyme by affinity chromatography

A relatively simple procedure is described for purifying human serum angiotensin-converting enzyme. The enzyme was purified 130,000-fold to electrophoretic homogeneity using affinity chromatography as the principal purification step. The ligand was an immobilized competitive inhibitor, d-cysteinyl-l-proline. A six-carbon spacer arm was satisfactory for trapping the enzyme; 80% of the bound enzyme was eluted with 3 m urea-1.0 m NaCl-0.1 m Tris, pH 8.3. The specific activity was 39 units/mg protein and the molecular weight (155,000), isoelectric point (4.7), kinetic properties, and the effect of various inhibitors are in agreement with published reports.     

Purification of streptokinase by affinity chromatography on immobilized acylated human plasminogen.

Streptokinase is an extracellular protein produced by several strains of streptococci. It functions in the species-specific conversion of plasminogen to plasmin. In this paper we describe the purification of streptokinase by affinity chromatography on human plasminogen acylated with p'-nitrophenyl p-guanidinobenzoate. The acylated and non-acylated plasminogen and plasmin were coupled to cyanogen bromide-activated Sepharose 4B and evaluated for streptokinase purification. These results show that a homogeneous preparation of streptokinase with high specific activity and high yield can be obtained using acylated plasminogen. This method permits the binding of one milligram of streptokinase per milliliter of swollen gel.     

Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography

Bovine seminal plasma contains three similar acidic proteins, which we have previously designated as BSP-A1, BSP-A2, and BSP-A3. These proteins contain two homologous domains that are similar to type II structures present in the gelatin-binding domain of fibronectin. The present data have revealed that these proteins, like fibronectin, also form complexes with gelatin, a denatured collagen. Based on this property, a single step affinity purification method has been developed. In addition to these three proteins BSP-A1, -A2 and -A3, another protein with an apparent molecular weight of 30,000 dalton (named BSP-30-kDa) also bound to the gelatin-agarose column. Elution of these proteins from affinity columns using a linear gradient of either urea or arginine gave essentially the same pattern with a high yield of 90–95%. The purified proteins were homogeneous by SDS-polyacrylamide gel electrophoresis, amino acid composition and HPLC. Chromatography of bull seminal vesicular fluid also exhibited an elution pattern similar to that obtained for bull seminal plasma. The availability of these purified proteins should aid in understanding the physiology of these gelatin-binding proteins.