Stabilization of fibroblast growth factors by a non-cytotoxic zwitterionic detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (chaps)

Abstract

The potential usefulness of a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHPAS), in the stabilization of acidic and basic fibroblast growth factors (FGFs) was examined. Among several detergents, CHAPS was found to be not only non-cytotoxic but also most useful in handling the diluted preparations of FGFs. The advantages are as follows: 1) at lower concentrations than 0.01% CHAPS did not affect growth factor activity of calf serum (CS) and the growth rate of BALB/c 3T3 cells. The primary culture of rat prostate epithelium and colony formation of NRK-49F cells were hardly influenced by CHAPS lower than 0.003%; 2) the loss of FGFs that usually occurs due to their adherence to the surface of storage containers was effectively prevented by inclusion of 0.1% CHAPS; 3) the recovery of FGFs after storage or dialysis was significantly enhanced by inclusion of 0.1% CHAPS; 4) CHAPS at lower concentrations than 0.1% does not interfere with amino acid analysis, except that Thr may be misled only when the ratio of protein/CHAPS is low; 5) amino acid sequence analysis was hardly disturbed by CHAPS up to 0.5%. These results indicate that CHAPS is useful as a stabilizing agent for various kinds of polypeptides capable of showing biological activity at a low concentration.

Monoclonal antibodies against heparin-binding growth factor II/basic fibroblast growth factor that block its biological activity: invalidity of the antibodies for tumor angiogenesis.

“…aFGF was further purified by cation-exchange HPLC on a Protein Pak G-SP column (8.2 x 75 mm) (Nihon Waters, Tokyo). The fractions eluted from the column with 0.35 M NaCI/50 mM sodium phosphate buffer (pH 6.8)/0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (20,21), which gave a single protein peak with DNA synthesis stimulating activity (see below), were collected. On SDS/19.5% polyacrylamide gel, the reduced samples of purified bFGF and aFGF gave single bands of protein with Mrs of 18,000 and 16,000, respectively.…”

“…Three peptides with sequences of 14-16 amino acids of the native form of bovine pituitary bFGF-(1-146) (8) and an additional tyrosine were synthesized by the solid-phase method in an automated Biolynx 4170 peptide synthesizer (Pharmacia LKB) or an automated Applied Biosystems 430 A peptide synthesizer (21).…”

“…Purified bovine bFGF was labeled with 1251 by the chloramine-T method as described by Kan et al (25) and purified by heparin-Sepharose affinity chromatography as described by Neufeld and Gospodarowicz (26) with some modifications. Briefly, 2.8 ,ug of bovine bFGF was incubated with 700 ,uCi (25.9 MBq) of 125I in the presence of 0.02% CHAPS (21), in 110 ,ul of reaction mixture containing 180 ,ug of chloramine T per ml for 2 min at room temperature. The reaction was stopped by adding 100 ,ul of 0.02 M dithiothreitol.…”

Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury

“…It has not been possible to quantitate the absolute amount ofbFGF that reached the heart as a substantial proportion adheres to the cannula and tubing [16].…”

Characterization of release of basic fibroblast growth factor from bovine retinal endothelial cells in monolayer cultures

“…At confluence the medium was removed and the monolayer washed with Hanks Balanced Salt Solution (HBSS). Then 2 ml of serum-free DMEM containing 0.01 % (w/v) CHAPS [27] was added to each flask, and the flasks were sealed and incubated at 37°C; 21 flasks were used for measuring cumulative growth-factor release, and 3 for measuring daily release from the same monolayer, and subsequent results and discussion will refer to either serum-free cumulative or daily release. Photographs were taken of the cells every day (Fig.…”