| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
There are some evidences showing that the bifunctional reagent glutaraldehyde (GA) might be a model compound suitable for an elucidation of post-translational modification of serum albumin. Since no one has reported the presence of GA in vivo, it is reasonable to assume that other unknown factors instead of GA may be involved in the formation of similar structure to modification with GA.In the present project, we tried to clarify the factor based on the affinity of polyclonal and monoclonal antibodies against GA-modified human serum albumin (pHSA).
First, we could successfully obtain the monoclonal antibody that does not react with HSA but reacts with pHSA.The antibody recognized not only pHSA but also serum albumins from other animals modified with GA, showing that the antibody recognized the haptenic determinant. Pyridinium ring formed by the reaction of GA with amino compound was considered as the haptenic structure. Next, BSA was incubated with various carbonyl compounds ; glycolaldehyde, glyceraldehyde, crotonaldehyde, malondialdehyde (MDA), methylglyoxal and glyoxal. Among them, BSAs modified with glyceraldehyde, glycolaldehyde and MDA were recognized by the antibody with the highest affinity. In order to elucidate the hapten structure, the reaction product of MDA with N-acetyl-glycyl-L-lysine methyl ester was separated based on the affinity of the antibody. Consequently, Fraction 1 was separated which has molecular weight of 699. Although the NMR analyses suggested the presence of heterocyclic structure in Fraction 1, the assignment is now in progress.
From these results, it was suggested that pHSA-like structure can be formed by the Maillard reaction of HSA with various carbonyl compounds in vivo and nitrogen-containing heterocyclic structure present in those reaction products may be involved in the binding with the antibody.
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