Development of in vitro model of metal-catalyzed oxidation of human serum albumin

Eric L. Kilpatrick, Lisa E. Kilpatrick, and David M. Bunk

Objective: Ischemically modified albumin (IMA) is currently used as an early ischemic indicator and is assayed by the in vitro measurement of cobalt binding to albumin which is reduced in the ischemic condition.  However, the specific chemical modifications which define IMA are as yet unknown and hinder further development of direct clinical measurement and standardization efforts.  The present study sought to investigate an in vitro model of metal-catalyzed oxidation (MCO) of human serum albumin (HSA) to mimic IMA and create an analytical methodology for the structural determination of modified albumin.  The four extreme amino terminus residues (DAHK) of HSA contain a high affinity cation binding site which has been reported to exhibit reduced in vitro binding of cobalt following exposure to ischemia in vivo.  Two proposed mechanisms to account for the reduced binding are modification of the binding residue side chains by MCO or competitive incorporation of copper at the binding site.  This study focused on the determination of structural modifications of MCO by using mass spectrometric techniques which are applicable to improved measurement methodologies.

Methods: Synthetic peptides corresponding to the N-terminal tryptic peptides of HSA (DAHK and DAHKSEVAHR), and intact HSA were subjected to MCO using the Fenton reaction with copper chloride, hydrogen peroxide and ascorbate.  The reaction products were analyzed by either matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI) or electrospray ionization tandem mass spectrometry (MS/MS).

Results: MALDI analysis of the DAHK synthetic peptide after MCO indicated no changes in its relative molecular masses.  However, a 16 Da shift was seen with the peptide DAHKSEVAHR after MCO.  The peptide oxidation products were also analyzed by direct infusion MS/MS revealing that the 16 Da shift occurred on the Histidine at position 9 and not at position 3, the proposed high affinity binding site.  Intact HSA subjected to MCO was digested by trypsin before analysis by LC/MS/MS.  The results of this analysis also revealed oxidation on the Histidine in position 9, consistent with the peptide results.

Conclusion: A methodology leading to the identification of the specific modifications of the amino-terminus of HSA caused by metal-catalyzed oxidation using a copper system are presented.  Specifically, experimental results indicate that the high affinity binding site, centered on the Histidine at position 3 was not found to be modified.  Surprisingly, the Histidine at position 9 was observed to be oxidized.  A consistent oxidation pattern was found in the in vitro MCO of both synthetic peptides and intact protein.  Likewise, similar results are seen utilizing two distinct mass spectrometric techniques; MALDI and LC/MS/MS.  The significant finding is that, in this scenario of MCO, it is not the high affinity binding site which is being modified, contrary to the current hypothesis of MCO of albumin.  While these results alone cannot be generalized to in vivo modification, they do provide clues to the nature of MCO and also provide a measurement methodology which, in future work, will be applied to the elucidation of the structure of in vivo ischemically modified albumin.

Eric Kilpatrick    Mentor: David Bunk

Analytical Chemistry Division

Chemical Sciences and Technology Laboratory

331 Fort Johnson Road

Charleston, SC 29412

telephone: 843-762-8830

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