ABSTRACT
A question of the existence of blood proteins binding unmodified insulin has remained open for many years. This paper is devoted to the description of a method of isolation and characteristics of the insulin-binding protein (IBP) isolated from rat and human blood sera. The level of IBP in the serum was 0.12-0.15 mg/ml. The protein was referred to IgG basing on the data of electrophoresis under denaturing conditions, immunoelectrophoresis and immunoenzymatic analysis. The constant of protein association with insulin varied with regard to a source within 1.5.10(-7) to 2.5.10(7) M-1. The studied protein bound both 125I-modified and unmodified insulin preparations. The calculation based on IBP parameters, showed that no less than half of the blood insulin must be in a bound condition. The capacity of such insulin-binding system is rather great and is capable of leveling down sharp changes in a blood insulin concentration.
Subject(s)
Blood Proteins/isolation & purification , Insulin/blood , Animals , Blood Protein Electrophoresis , Blood Proteins/metabolism , Chromatography, Affinity , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Humans , Immunoelectrophoresis , Immunoglobulin G/isolation & purification , Immunoglobulin G/metabolism , Molecular Weight , Protein Binding , RatsABSTRACT
Two modifications of solid phase enzyme immunoassay (EIAA) for insulin with the use of beta-lactamase from B. licheniformis 749/c and horse radish peroxidase as the markers were developed. beta-Lactamase and peroxidase conjugates with insulin were prepared with the glutaric aldehyde and periodate methods respectively. Both the conjugates were stable and preserved high immunospecific and enzymatic activity. Sensitivity of EIAA with the use of the beta-lactamase or peroxidase as the markers was the same and amounted to 15 ng/ml of insulin. However, with the use of the beta-lactamase in EIAA not only spectrophotometric recording but also visual registration of the results was possible.
