Infrared spectroscopic studies of time-dependent changes in fibrinogen adsorbed to polyurethanes

Lenk, T. J.; Horbett, Thomas A.; Ratner, Buddy D.; Chittur, Krishnan K.

doi:10.1021/la00056a030

Cited by 104 publications

(97 citation statements)

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“…In peptides and proteins the intensity ratio of the amide I/II band is reported to show high sensitivity to backbone orientation in films or membrane that can also be affected by conformational perturbation and experimental methodology for example, transmission or reflectance optics employed for spectral acquisition. [20,21] Since the amide I/II intensity ratios of the SC/KBr pellet analyzed using transmission FTIR and DRIFT are identical we conclude these differences are not due to the difference in experimental methods. This led us to address the question of whether subjecting protein to pressure compression during the KBr pellet formation affects the structural stability.…”

Section: Diffuse Reflectance Infrared Fourier Transform Spectroscopymentioning

confidence: 71%

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

et al. 2009

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Structure of immobilised enzymes: Circular dichroism, infrared and fluorescence spectroscopic methods are used to characterise in situ structural stability of enzymes immobilised on particles. The enzyme subtilisin Carlsberg exhibits a similar secondary structure in solution and in the immobilised state but on the surface of porous silica gel a rigid tertiary structure is preferred (see picture). We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carlsberg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435). SC shows nearly identical secondary structure in solution and in the immobilised state as evident from far UV CD spectra and amide I vibration bands. Increased near UV CD intensity and reduced Trp fluorescence suggest a more rigid tertiary structure on the silica surface. After immobilised SC is inactivated, these techniques reveal: a) almost complete loss of near UV CD signal, suggesting loss of tertiary structure; b) a shift in the amide I vibrational band from 1658 cm−1 to 1632 cm−1, indicating a shift from α‐helical structure to β‐sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to β‐sheet structure; d) strong increase in Trp fluorescence intensity, which reflects reduced intramolecular quenching with loss of tertiary structure; and e) major change in fluorescence lifetime distribution, confirming a substantial change in Trp environment. DRIFT measurements suggest that pressing KBr discs may perturb protein structure. With the enzyme on organic polymer it was possible to obtain near UV CD spectra free of interference by the carrier material. However, far UV CD, DRIFT and fluorescence measurements showed strong signals from the organic support. In conclusion, the spectroscopic methods described here provide structural information hitherto inaccessible, with their applicability limited by interference from, rather than the particulate nature of, the support material.

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Section: Diffuse Reflectance Infrared Fourier Transform Spectroscopymentioning

confidence: 71%

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

et al. 2009

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“…For example, they analyze the ratio of the amide I to amide II band intensity, which is known to be related to the orientation of the protein amides with respect to the surface. [39,40] In all their experiments, they obtain an amide I to amide II ratio near 1.0. They conclude that the orientation of the protein does not change.…”

Section: Discussionmentioning

confidence: 92%

Measuring hydrogen–deuterium exchange in protein monolayers

Smith

Cicerone

Meuse

2009

Surface & Interface Analysis

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A Fourier transform infrared (FTIR) spectroscopy assay to measure hydrogen-deuterium exchange (HDX) in surface-adsorbed protein monolayers is developed to provide information on protein tertiary structure, because the typical secondary structural analysis of our surface and solution protein samples proved to be very similar. Adsorbed protein HDX is quantified by exposing the protein to a 50% deuterated NaPO 4 buffer solution and then measuring the normalized intensity change of the amide II band in the FTIR reflection spectrum. When collected as a function of exchange time, this intensity follows the kinetics of the exposure of the protein amides to solvent. HDX kinetics have been obtained for bovine serum albumin (BSA) in solution and adsorbed to gold surfaces. Using experiments designed to allow comparisons between protein in solution and on surfaces, the extent of HDX was found to increase over that observed for BSA in solution, consistent with an increase in the exposure of albumin amide groups and protein unfolding upon adsorption. We also show that BSA adsorbs to the surface of gold in multilayers and that the increase in amide exposure is present only in the first adsorbed monolayer. Published in

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“…The disks then were thoroughly rinsed with sterile PBS and, in some cases, then incubated for one additional h with 1% SDS solution with mixing at 80 rpm (Orbital shaker, Hoefer Scientific Instruments, San Francisco, CA) in order to remove elutable (nondenatured) fibrinogen. 34 The surface-associated radioactivity on disks rinsed with PBS and 1% SDS was taken as measurements of adsorbed and ''denatured'' fibrinogen, respectively.…”

Section: Measurement Of Adsorbed and ''Denatured'' Fibrinogenmentioning

confidence: 99%

Fibrinogen adsorption and host tissue responses to plasma functionalized surfaces

Tang

Timmons

1998

J. Biomed. Mater. Res.

110

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The physical and chemical characteristics of material surfaces are thought to play important roles in biomaterial-mediated tissue responses. To understand the importance of discrete biomaterial chemical characteristics in modifying host tissue responses, we constructed surfaces bearing different functional groups using radio frequency glow discharge plasma polymerization. Surfaces evaluated included those having high concentrations of -OH, -NH2, -CF3, and siloxyl groups. These surfaces and polyethylene terephthalate controls were used to assess the importance of particular physicochemical characteristics in surface:protein:cell interactions both in vitro and in vivo. The results obtained show that surface functionalities do significantly affect both the adsorption and "denaturation" of adsorbed fibrinogen (which is an important mediator of inflammatory responses to biomaterial implants). In addition, these surfaces provoke different degrees of acute inflammatory responses. Interestingly, the amounts of "denatured" fibrinogen that spontaneously accumulate on the individual surfaces correlate closely with the extent of biomaterial-mediated inflammation. These results suggest that surfaces that tend to "irreversibly" bind fibrinogen prompt greater acute inflammatory responses. Unexpectedly, all test surfaces except those bearing a siloxyl group engender relatively similar biomaterial-mediated fibrotic responses. Thus surface functionalities alone may not be sufficient to affect subsequent fibrotic responses.

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Infrared spectroscopic studies of time-dependent changes in fibrinogen adsorbed to polyurethanes

Cited by 104 publications

References 0 publications

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

Measuring hydrogen–deuterium exchange in protein monolayers

Fibrinogen adsorption and host tissue responses to plasma functionalized surfaces

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