Pollen grain column chromatography: quantitation and biochemical analysis of ragweed-pollen solutes.
The kinetics, quantitative yield, and sequence of solute release during the extraction of allergenic substances from short ragweed (Ambrosia artemisiifolia) pollen were compared with a conventional batch-type method and the novel technique of pollen grain column chromatography. With the batch method, 14.6 +/- 1.7 mg of pollen solutes were eluted per 100 mg of dried defatted pollen in 1 minute; the 24-hour solute yield was 27.4 +/- 2.7 mg. With the column method, 3.7 +/- 1.3 mg of pollen solutes were eluted in 1 minute; the 24-hour solute yield was 29.3 +/- 2.1 mg. The kinetics of solute release with the column method were modeled as the simultaneous first-order elution of ragweed-pollen solutes into three hypothetical compartments. The theoretical initial solute concentration was 50 gm/L. The isoelectric focusing patterns, optical properties, distributions of enzymes, Ra5, and antigen E activities were consistent with the sequential separation of ragweed-pollen solutes and the three compartment model. Enzyme activities were eluted either maximally in the first minute (phosphatases and N-acetyl-beta-glucaminidase) or delayed until 10 minutes (leucine aminopeptidase). Ra5 was eluted rapidly, whereas antigen E was eluted during a more prolonged period. Pollen grain chromatography provides a simple, reproducible method for studying pollen solute release.
Baraniuk
J N
JN
Department of Medicine, Duke University, Durham, NC 27710.
Esch
R E
RE
Buckley
C E
CE
D000135:Acid Phosphatase / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000485:Allergens; D052179:Antigens, Plant; D002457:Cell Extracts / Q000032:analysis; D002845:Chromatography / Q000379:methods*; D004950:Esterases / Q000378:metabolism; D007525:Isoelectric Focusing; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010940:Plant Proteins; D011058:Pollen / Q000378:metabolism; D013379:Substrate Specificity
D000485:Allergens; C026967:Amb a I protein, Ambrosia artemisiifolia; D052179:Antigens, Plant; D002457:Cell Extracts; D010940:Plant Proteins; C069543:group 5 allergens, Ambrosia; D004950:Esterases; D000469:Alkaline Phosphatase; D000135:Acid Phosphatase; D010744:Phosphoric Monoester Hydrolases