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Separation of complexes containing protein A and IgG or Fc gamma fragments by high-performance liquid chromatography

Journal Article · · Anal. Biochem.; (United States)
; ;

Protein A of Staphylococcus aureus is a bivalent Fc receptor that can form complexes with immunoglobulin G (IgG) or Fc gamma fragments that activate humoral (e.g., complement) and cellular (e.g., lymphocyte) components of the immune system both in vitro and in vivo. To obtain complexes formed between protein A of Staphylococcus aureus (SpA) and rabbit IgG or Fc gamma fragments for purposes of characterizing their compositions and studying their biological activities, the authors have used high-performance liquid chromatography to separate complexes in 20 min. Complexes were prepared with trace amounts of /sup 125/I-SpA and /sup 131/I-IgG or /sup 131/I-Fc gamma to simplify the analyses. With excess molar amounts of IgG or Fc gamma the complexes have the molecular formulas ((IgG)2SpA)2 or ((Fc gamma)2SpA)2. With excess SpA, complexes corresponding to (IgG)(SpA) or (Fc gamma)(SpA) are formed, perhaps with other complexes that have different ratios of components. Since SpA is a rod-shaped molecule it elutes at a molecular weight corresponding to 240,000 rather than the true value of 42,000. This behavior is reflected in the elution of certain complexes at shorter retention times than expected on the basis of actual molecular weights, and facilitates separation of complexes from free IgG or Fc gamma. The true molecular weights and molecular formulas of complexes isolated by HPLC were verified by ultracentrifugation. This HPLC method was used to study the interconversion and stability of complexes.

OSTI ID:
5016814
Journal Information:
Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 1; ISSN ANBCA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
ANIMALS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CENTRIFUGATION
CHROMATOGRAPHY
COMPLEMENT
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
GLOBULINS
IMMUNOGLOBULINS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE 131
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LEUKOCYTES
LIQUID COLUMN CHROMATOGRAPHY
LYMPHOCYTES
MAMMALS
MATERIALS
MICROORGANISMS
MOLECULAR WEIGHT
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PROTEINS
RABBITS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
SEPARATION PROCESSES
SOMATIC CELLS
STAPHYLOCOCCUS
TRACER TECHNIQUES
ULTRACENTRIFUGATION
VALENCE
VERTEBRATES