Raster image correlation spectroscopy as a novel tool for the quantitative assessment of protein diffusional behaviour in solution.

Abstract:

:The application of raster image correlation spectroscopy (RICS) as a tool for the characterisation of protein diffusion was assessed using a model protein, bovine serum albumin (BSA), as a function of formulation and denaturing conditions. RICS results were also validated against dynamic light scattering and fluorescence correlation spectroscopy. Results from this study demonstrate correlation between outputs obtained from the three experimental techniques. Ionic strength independency was observed at pH 7, and a reduction in the corresponding diffusion coefficients was noted at pH 4.5 for 1 µM BSA-Alexa Fluor 488. Conversely, at pH 5.2, higher-concentration samples exhibited ionic strength dependency. Buffer composition, sample pretreatment, thermal denaturation and freeze-thaw cycling were also found to influence RICS output, with a reduction in the diffusion coefficient and the number of particles observed for both pH values. In conclusion, RICS analysis of images acquired using a commercial confocal microscope offers a potential scope for application to both quantitative and qualitative characterisation of macromolecular behaviour in solution.

journal_name

J Pharm Sci

authors

Hamrang Z,Pluen A,Zindy E,Clarke D

doi

10.1002/jps.23105

subject

Has Abstract

pub_date

2012-06-01 00:00:00

pages

2082-93

issue

6

eissn

0022-3549

issn

1520-6017

pii

S0022-3549(15)31570-7

journal_volume

101

pub_type

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