Affinity chromatography of thyroxine-binding proteins from human serum

The affinity matrix prepared by the attachment of L-thyroxine (T4) to epichlorohydrine-activated Sepharose 4B biospecifically absorbs the T4-binding globulin (TBG), 25K and 80/27K proteins, immunoglobulin G (IgG) and albumin (HSA) from human normal and retroplacental sera. The absorbed protein patterns were shown to depend on the immobilized T4 concentration, pH, temperature and incubation time. The potent eluents desorbing 85-100% of the protein are 1 mM NaOH, 3 M NH4SCN, 10(-5) M T4 or 3 mM 8-anilinonaphthalene-1-sulfonic acid (ANS) for TBG; NaOH, NH4SCN, 3 mM MgCl2 or 12mM sodium cholate for 25K protein and HSA; NaOH, NH4SCN or MgCl2 for the 80/27K and 25K proteins and IgG. Moreover, T4 desorbs small amounts (6-8%) of the 80/27K and 25K proteins, while sodium cholate elutes about 6% of TBG. The eluted from T4-Sepharose 4B and further purified TBG, 25K and 80/27K proteins display different [125I]T4-binding activities within the pH range from 2 to 9 and differ by their resistance to thermal inactivation at 50-80 degrees C. Double radial immunodiffusion analysis with the use of antisera to TBG, 25K, 80/27K, HSA and IgG demonstrated that the proteins share no common antigenic determinants. It was concluded that the novel 25K and 80/27K proteins represent endogenous components of the human blood thyroid hormone-binding protein system rather than fragments or aggregates of the known T4-binding proteins.     

Thiophilic adsorption chromatography: the separation of serum proteins

Human serum proteins were separated on a matrix obtained by reaction of beta-mercaptoethanol with divinyl sulfone-activated agarose (the so-called T-gel). Binding of almost all serum proteins was observed at high concentrations of ammonium sulfate. Elution was achieved by gradually lowering the concentration of salt in the washing buffer. Fractions obtained during elution were analyzed by fused rocket immunoelectrophoresis. Proteins were recovered in high yields and with an excellent separation in this one-step procedure ("Thiophilic adsorption chromatography"). A rapid and straightforward procedure giving essentially pure immunoglobulins from crude rabbit serum with at least 80% yield by the T-gel is also presented.     

Ion-exchange displacement chromatography of serum proteins, using carboxymethyldextrans as displacers

A system of displacers comprising carboxymethyldextrans with progressively higher content of carboxyl groups forms a displacement train in which absorbed proteins find positions according to their affinities for the adsorbent, an anion exchanger. Because little or no salt need be used, effluent fractions can be evaluated directly by gel electrophoresis. Application to the fractionation of serum proteins is demonstrated.     

Affinity chromatography of serum proteins using immobilized lectin from Vicia faba

When human serum is applied to a column of Sepharose-insolubilized lectin from Vicia faba, some serum proteins are bound which can be eluted by means of 0.1 M glucose solution. These proteins are parts of the immunoglobulins IgA, IgG, IgM, and the alpha2-macroglobulin. These particular types of serum protein are bound specifically, due perhaps to some structural variation in the carbohydrate moieties they contain.     

Analysis of lidocaine interactions with serum proteins using high-performance affinity chromatography

High-performance affinity chromatography was used to study binding by the drug lidocaine to human serum albumin (HSA) and α₁-acid glycoprotein (AGP). AGP had strong binding to lidocaine, with an association equilibrium constant (K_a) of 1.1–1.7 × 10⁵ M^?1 at 37 °C and pH 7.4. Lidocaine had weak to moderate binding to HSA, with a K_a in the range of 10³ to 10⁴ M^?1. Competitive experiments with site selective probes showed that lidocaine was interacting with Sudlow site II of HSA and the propranolol site of AGP. These results agree with previous observations in the literature and provide a better quantitative understanding of how lidocaine binds to these serum proteins and is transported in the circulation. This study also demonstrates how HPAC can be used to examine the binding of a drug with multiple serum proteins and provide detailed information on the interaction sites and equilibrium constants that are involved in such processes.     

Affinity chromatography of human serum proteins using immobilized lectin from Allomyrina dichotoma

The beta-D-galactoside-specific lectin from Allomyrina dichotoma reacts with serum proteins which contain the corresponding carbohydrate moieties. By affinity chromatography of human serum using the insolubilized lectin coupled to Sepharose, it is possible to fractionate human serum proteins in 2 groups: those which react with the lectin (alpha 1-acid glycoprotein, haptoglobin, etc.) and those which do not (albumin, Gc-globulin, etc.). IgG is the only serum protein that can be found in both groups.     

Direct serum injection in micellar liquid chromatography : Recovery of serum proteins and assay of hydrophilic drugs

The recovery of serum proteins from reversed-phase and internal-surface reversed-phase (ISRP) silica supports following direct serum injection was investigated using an eluent containing a micellar solution of sodium dodecyl sulphate (SDS). The results indicated that the recoveries of serum proteins were 98–103% for both supports. On the basis of the above findings, the separation and recovery of hydrophilic drugs (cephalosporins and salicylic acid) from human serum were investigated using acidic eluents including micellar solutions of SDS. They were completely separated from the components of serum, and the recoveries were 94–98% despite protein binding. Although the recommended eluent pH range is 6.0–7.5 for the ISRP support, eluents of pH 2–8 can be used with the micellar chromatographic system.     

The myostatin propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum

Myostatin, also known as growth and differentiation factor 8, is a member of the transforming growth factor beta superfamily that negatively regulates skeletal muscle mass (1). Recent experiments have shown that myostatin activity is detected in serum by a reporter gene assay only after activation by acid, suggesting that native myostatin circulates as a latent complex (2). We have used a monoclonal myostatin antibody, JA16, to isolate the native myostatin complex from normal mouse and human serum. Analysis by mass spectrometry and Western blot shows that circulating myostatin is bound to at least two major proteins, the myostatin propeptide and the follistatin-related gene (FLRG). The myostatin propeptide is known to bind and inhibit myostatin in vitro (3). Here we show that this interaction is relevant in vivo, with a majority (>70%) of myostatin in serum bound to its propeptide. Studies with recombinant V5-His-tagged FLRG protein confirm a direct interaction between mature myostatin and FLRG. Functional studies show that FLRG inhibits myostatin activity in a reporter gene assay. These experiments suggest that the myostatin propeptide and FLRG are major negative regulators of myostatin in vivo.