title: Characterization of protein immobilization at silver surfaces by near edge X-ray absorption fine structure spectroscopy.
abstract: Ribonuclease A (RNase A) is immobilized on silver surfaces in oriented and random form via self-assembled monolayers (SAMs) of alkanethiols. The immobilization process is characterized step-by-step using chemically selective near-edge X-ray absorption fine structure spectroscopy (NEXAFS) at the C, N, and S K-edges. Causes of imperfect immobilization are pinpointed, such as oxidation and partial desorption of the alkanethiol SAMs and incomplete coverage. The orientation of the protein layer manifests itself in an 18% polarization dependence of the NEXAFS signal from the N 1s to pi* transition of the peptide bond, which is not seen for a random orientation. The S 1s to C-S sigma* transition exhibits an even larger polarization dependence of 41%, which is reduced to 5% for a random orientation. A quantitative model is developed that explains the sign and magnitude of the polarization dependence at both edges. The results demonstrate that NEXAFS is able to characterize surface reactions during the immobilization of proteins and to provide insight into their orientations on surfaces.
authors.affiliation: Departments of Physics, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
authors.forename: Xiaosong
authors.initials: X
authors.lastname: Liu
authors.forename: Chang-Hyun
authors.initials: CH
authors.lastname: Jang
authors.forename: Fan
authors.initials: F
authors.lastname: Zheng
authors.forename: Astrid
authors.initials: A
authors.lastname: Jürgensen
authors.forename: J D
authors.initials: JD
authors.lastname: Denlinger
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Dickson
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Raines
authors.forename: Nicholas L
authors.initials: NL
authors.lastname: Abbott
authors.forename: F J
authors.initials: FJ
authors.lastname: Himpsel
mesh_terms: D000042:Absorption; D002244:Carbon / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D015394:Molecular Structure; D009584:Nitrogen / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism; D012834:Silver / Q000737:chemistry*; D013053:Spectrophotometry; D013455:Sulfur / Q000737:chemistry; D013499:Surface Properties; D014965:X-Rays
chemical_list: D004800:Enzymes, Immobilized; D010455:Peptides; D012834:Silver; D013455:Sulfur; D002244:Carbon; D012260:Ribonucleases; D009584:Nitrogen