Non-specific protein binding by adsorbents designed for the specific affinity chromatography of sialidase in crude bacterial extracts

Abstract

A number of derivatives of Sepharose 4B gels were prepared containing tyramine,-Gly-Gly-Tyr and the tetrapeptide, Thr (But)-Phe-Pro-Tyr-. A specific inhibitor of sialidase, p-diazo-phenyl oxamic acid, was also coupled to these phenolsubstituted Sepharose gels. The effectiveness of these gels to purify sialidase by "affinity" chromatography was examined. The sialidase from Vibrio cholerae was adsorbed by these gels and subsequently re-eluted by a change in pH and temperature. The adsorption and elution of enzyme was independent of the presence or absence of the "specific inhibitor" of the sialidase, phenyl oxamic acid. A mechanism for the adsorption and subsequent elution of the sialidase is proposed. It is important to re-evaluate the biological data obtained with sialidase prepared by the affinity column recommended by Cuatrecasas and Illiano, Biochem. Biophys. Res. Commun. 44, 178-184 (1971), since glycosidases from Clostridium perfringens other than sialidase are also adsorbed and eluted under the conditions specified.

Purification and Characterization of Blood Group A-degrading Isoforms of α-N-Acetylgalactosaminidase from Ruminococcus torques Strain IX-70

“…But C. perfringens strains also produce toxins and other extracellular hydrolases which make purification of a single glycosidase difficult (5,11,12), whereas ␣-GalNAcases from nonbacterial sources have acidic pH optima in the range 3.5-4.8 and little or no activity at pH Ն7.0 (liver (10,(13)(14)(15)(16)(17)(18)(19)(20), fungi (21,22), mollusks (23)(24)(25)(26)(27), and Ehrlich ascites tumor cells (28)). For these reasons we purified ␣-GalNAcase from the culture supernatants of Ruminococcus torques strain IX-70 (ATCC 35915) and report here on the purification and properties of three of its isoforms.…”

Neuraminidase and Hematopoietic Factors from Human Urine

“…One method, heat treat ment, common for inactivating enzymes [29,30], eliminated a maximum of 80% of en zyme activity without loss of hematopoietic activity. Second, oxamic acid gel chromatog raphy, which absorbs certain kinds of neur aminidases, revealed enzyme activity in the column effluent, implying only partial ab sorption [31]. In addition, lectin-Sepharose chromatographies, including Concanavalin-A and ion exchange chromatographies (DEAE cellulose, CM cellulose, and SP-Sephadex), did not permit clear separation of neuraminidase from hematopoietic factors.…”

Short Communications. An improved method for purifying sialidase

“…This adsorbent rontains a sialidase inhibitor of as yet uncertain action (an Nsubstituted oxamnic acid) lined to Sepharose by a tripeptide spacer arm, The adsorption of sialidase and contaminating proteins to this material appears to be non-specific. There is, bowever, some uncertainty as to whether the adsorption arises from ion-exchange phenomena (Rood & Wilkinson, 1974) or hydrophobic effects (Huang & Aminoff, 1974).…”