Improved assay for measuring heparin binding to bull sperm

Journal Article · Fri Jan 01 00:00:00 EST 1988 · J. Dairy Sci.; (United States)

DOI:https://doi.org/10.3168/jds.S0022-0302(88)79547-8· OSTI ID:5244252

Miller, D J; Ax, R L

The binding of heparin to sperm has been used to study capacitation and to rank relative fertility of bulls. Previous binding assays were laborious, used 10/sup 7/ sperm per assay point, and required large amounts of radiolabeled heparin. A modified heparin-binding assay is described that used only 5 x 10/sup 4/ cells per incubation well and required reduced amounts of (/sup 3/H) heparin. The assay was performed in 96-well Millititer plates, enabling easy incubation and filtering. Dissociation constants and concentrations of binding sites did not differ if analyzed by Scatchard plots, Woolf plots, or by log-logit transformed weighted nonlinear least squares regression, except in the case of outliers. In such cases, Scatchard analysis was more sensitive to outliers. Nonspecific binding was insignificant using nonlinear logistic fit regression and a proportion graph. The effects were tested of multiple free-thawing of sperm in either a commercial egg yolk extender, 40 mM Tris buffer with 8% glycerol, or 40 mM Tris buffer without glycerol. Freeze-thawing in extender did not affect the dissociation constant or the concentration of binding sites. However, freeze-thawing three times in 40 mM Tris reduced the concentration of binding sites and lowered the dissociation constant (raised the affinity). The inclusion of glycerol in the 40 mM Tris did not significantly affect the estimated dissociation constant or the concentration of binding sites as compared to 40 mM Tris without glycerol.

• Research Organization: Univ. of Wisconsin, Madison, WI (United States)
• OSTI ID: 5244252
• Journal Information: J. Dairy Sci.; (United States), Vol. 71:1
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
HEPARIN
BIOCHEMICAL REACTION KINETICS
RADIOASSAY
SPERMATOZOA
AFFINITY
CATTLE
FERTILITY
MEASURING METHODS
TRITIUM COMPOUNDS
AMINES
ANIMALS
ANTICOAGULANTS
CARBOHYDRATES
DOMESTIC ANIMALS
DRUGS
GAMETES
GERM CELLS
HEMATOLOGIC AGENTS
KINETICS
LABELLED COMPOUNDS
MAMMALS
MUCOPOLYSACCHARIDES
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
POLYSACCHARIDES
REACTION KINETICS
RUMINANTS
SACCHARIDES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques