RESUMO
Human serum albumin-Sepharose was prepared by coupling human serum albumin to cyanogen bromide activated Sepharose 4B. This immunoadsorbent showed considerable non-specific protein adsorption. The adsorbed proteins were mainly immunoglobulins which could not be separated from required antibody. It is suggested that basic groups formed in the preparation of the albumin-Sepharose are responsible for this non-specific protein adsorption. Non-specific protein adsorption could be completely eliminated by neutralizing the basic groups of the albumin-Sepharose with the anionic dye blue dextran. The resultant blue dextran-albumin-Sepharose allowed purification of anti-albumin-antibody with high yield. It is shown that the isolated antibody is pure and retains its native properties.
Assuntos
Especificidade de Anticorpos , Polissacarídeos , Sefarose , Albumina Sérica/imunologia , Adsorção , Anticorpos/análise , Anticorpos/isolamento & purificação , Humanos , Ligação ProteicaRESUMO
Biosynthetic threonine deaminase (L-threonine hydro-lyase (deaminating), EC 4.2.1.16) was purified to apparent homogeneity from cell extracts of Escherichia coli by chromatographic procedures using valine-Sepharose, isoleucine-N-hexamethyleneamine-Sepharose, and hydroxyapatite with an overall yield of 40%. Analytical ultracentrifugation shows a molecular weight of 214 000. In sodium dodecyl sulfate gel electrophoresis, the enzyme migrates as a single band corresponding to a molecular weight of about 50 000. These data confirm that the enzyme is a tetramer. The sedimentation coefficient, s-020,w, determined by differential sedimentation experiments is 9.2 S. The enzyme shows absorption maxima at 415 and 280 nm. Determination of pyridoxal phosphate by three indenpendent methods shows the presence of two molecules of pyridoxal phosphate per enzyme molecule, the different methods being in excellent agreement equilibrium dialysis experiments establish the presence of two isoleucine binding sites. The Scatchard plot suggests non-cooperativity of these sites. The association constant for isoleucine is 1.2 - 10(5)M-1.
