On the distribution of protein refractive index increments

The protein refractive index increment, dn/dc, is an important parameter underlying the concentration determination and the biophysical characterization of proteins and protein complexes in many techniques. In this study, we examine the widely used assumption that most proteins have dn/dc values in a very narrow range, and reappraise the prediction of dn/dc of unmodified proteins based on their amino acid composition. Applying this approach in large scale to the entire set of known and predicted human proteins, we obtain, for the first time, to our knowledge, an estimate of the full distribution of protein dn/dc values. The distribution is close to Gaussian with a mean of 0.190 ml/g (for unmodified proteins at 589 nm) and a standard deviation of 0.003 ml/g. However, small proteins <10 kDa exhibit a larger spread, and almost 3000 proteins have values deviating by more than two standard deviations from the mean. Due to the widespread availability of protein sequences and the potential for outliers, the compositional prediction should be convenient and provide greater accuracy than an average consensus value for all proteins. We discuss how this approach should be particularly valuable for certain protein classes where a high dn/dc is coincidental to structural features, or may be functionally relevant such as in proteins of the eye.     

Cell nuclei have lower refractive index and mass density than cytoplasm

Common perception regards the nucleus as a densely packed object with higher refractive index (RI) and mass density than the surrounding cytoplasm. Here, the volume of isolated nuclei is systematically varied by electrostatic and osmotic conditions as well as drug treatments that modify chromatin conformation. The refractive index and dry mass of isolated nuclei is derived from quantitative phase measurements using digital holographic microscopy (DHM). Surprisingly, the cell nucleus is found to have a lower RI and mass density than the cytoplasm in four different cell lines and throughout the cell cycle. This result has important implications for conceptualizing light tissue interactions as well as biological processes in cells.

     

Buffer dependence of refractive index increments of protein solutions

The refractive index increment of a protein solution is a property not only of the protein, but also of the solvent. This is demonstrated theoretically and confirmed experimentally using analytical interferometry. © 1998 John Wiley & Sons, Inc. Biopoly 46: 489–492, 1998     

Estimation of the size of bilayer liposome by optical density and refractive index

A simple method for estimating a mean particles size in a suspension is suggested on the basis of an empirical correlation of a liposome size found by the optical shift method and on optic properties of the suspension. For liposome size characteristics only two parameters: optical density and refraction index increment are measured.     

Determination of protein absorption coefficients using a refractive index chromatographic monitor

Using lysozyme as a primary standard, a refractive index monitor designed for column chromatography was used to determined protein concentration and hence absorption coefficients. the method is non-destructive, requires only small amounts of protein 0.2 mg, and could be adapted for smaller samples.     

Concentration of protein in fibrin fibers and fibrinogen polymers determined by refractive index matching

We have used refractive index matching to determine the concentration of protein in the fibers in fibrin clots and of needlelike crystals of native fibrinogen. Our results are in agreement with those of Carr and Hermans [(1978) Macromolecules 11 , 46–50], as determined by light scattering—namely, that protein makes up about 20% of the volume of the fiber. However, we have found that the protein concentration is strongly dependent on ionic strength. An increase in ionic strength caused a substantial drop in the protein concentration. In a buffer containing 100 mM NaCl, the protein concentration was 26.6–29.8 g of protein per 100 cm³ of polymer, and at 200 mM NaCl it was reduced to 22.1–23.1 g/100 cm³.     

Deoxyribonueleate solutions: Sedimentation in a density gradient,partial specific volumes,density and refractive index increments,and preferential interactions

Density increments (?ρ/?c₂)°_μ in solutions of NaDNA in NaCl and CsDNA in CsCl were determined over a wide range of salt concentrations; calf thymus DNA, fragmented by sonic irradiation to a molecular weight of 4–6 × 10⁵ was used. The partial specific volume v?₂° of NaDNA at 25°C was found to ho 0.500 ml/g in water, and that of CsDNA 0.440 ml/g. Both values increase with increasing NaCl and CsCl concentration. Refractive index increments under various experimental conditions were also determined. The relevance of the density increments (at constant, chemical potential of diffusible solutes) to equilibrium sedimentation in a density gradient and the evaluation of molecular weights is discussed. Distribution coefficients of diffusible components, sometimes referred to as preferential solvation or net hydration, were derived from the density increments and partial volumes and compared with direct experimental results, whenever available, from membrane distribution and isopiestic distillation. The thermo-dynamic significance of the distribution coefficients as well as possible interpretations in terms of specific molecular mechanisms are considered.     

Laser interferometry of the hydrolytic changes in protein solutions: the refractive index and hydration shells

Using an original laser interferometer of enhanced sensitivity, an increase in the refractive index of a protein solution was observed during the reaction of proteolysis catalyzed by pepsin. The increase in the refractive index of the protein solution at a concentration of 4 mg/ml was \( 9 \times 10^{-6} \) for bovine serum albumin and \(2.4 \times 10^{- 6}\) for lysozyme. The observed effect disproves the existing idea that the refractive index of protein solutions is determined only by their amino acid composition and concentration. It is shown that the refractive index also depends on the state of protein fragmentation. A mathematical model of proteolysis and a real-time method for estimating the state of protein hydration based on the measurement of refractive index during the reaction are proposed. A good agreement between the experimental and calculated time dependences of the refractive index shows that the growth of the surface of protein fragments and the change in the number of hydration cavities during proteolysis can be responsible for the observed effect.     

Imaging cells and tissues with refractive index radiology

Can individual cells, including live cells, be imaged using hard x rays? Common wisdom until now required sophisticated staining techniques for this task. We show instead that individual cells and cell details can be detected in culture solution and tissues with no staining and no other contrast-enhancing preparation. The sample examined can be much thicker than for many other microscopy techniques without sacrificing the capability to resolve cells. The key factor in our approach is the use of a coherent synchrotron source and of contrast mechanisms based on the refractive index. The first successful tests were conducted on a variety of cell systems including skin and internal leaf cells, mouse neurons, rabbit fibroblast cells, and human tumor cells.     

Miscibility studies of HPMC/PEG blends in water by viscosity,density, refractive index and ultrasonic velocity method

Hydroxy propyl methyl cellulose (HPMC)/polyethylene glycol (PEG) blends are edible polymer films used for food packing and directly in foodstuffs. However, they are water-soluble in ordinary temperature and have good mechanical properties. The miscibility of HPMC/PEG blend in water was studied by viscosity, ultrasonic velocity, density and refractive index techniques at 30 and 50 °C. Using viscosity data, the interaction parameters μ and α were calculated. These values revealed that HPMC/PEG blend is miscible when the HPMC content is more than 60 wt.% in the blend at 30 and 50 °C, below which is immiscible. Further the result was also confirmed by ultrasonic velocity, density, refractive index measurements, which also revealed that the change in temperature has no significant effect on the miscibility of HPMC/PEG polymer blend.     

Miscibility studies of HPMC/PVA blends in water by viscosity, density, refractive index and ultrasonic velocity method

Hydroxy propyl methyl cellulose (HPMC)/polyvinyl alcohol (PVA) blends are edible polymer films used for food packing and directly in foodstuffs. However they are water-soluble in ordinary temperature and have good mechanical properties. The miscibility of HPMC/PVA blend in water was studied by viscosity, ultrasonic velocity, density and refractive index techniques at 30 and 50 °C. Using viscosity data, the interaction parameters μ and α were calculated. These values revealed that HPMC/PVA blend is miscible when the HPMC content is more than 60% in the blend at 30 and 50 °C. And also the result revealed that the change in temperature has no significant effect on the miscibility of HPMC/PVA polymer blend.     

Time-resolved refractive index change during the bacteriorhodopsin photocycle

Kinetic refractive index spectroscopy has been applied to the study of the bacteriorhodopsin photocycle. A fully hydrated purple membrane film was examined in the temperature range from 10° to 40°C using 532 nm excitation (doubled Nd YAG laser) and 633 nm (He–Ne laser) testing beam. Multiexponential fitting of the data revealed five processes. Four of them are well known from kinetic optical absorption studies. The fifth process has only recently been observed in optical absorption experiments where it has a relatively small amplitude. In our refractive index experiments it has an amplitude of up to 30% of the full signal amplitude. It is characterized by an Arrhenius temperature dependence with an activation enthalpy of 40±5 kJ/mol and a decay time of about 0.8 ms at 20°C.     

Bacterial infection of macrophages induces decrease in refractive index

Infection of cells by pathogens leads to both biochemical and structural modifications of the host cell. To study the structural modifications in a label‐free manner, we use digital holographic microscopy, DHM, to obtain the integral refractive index distribution of cells. Primary murine bone marrow derived macrophages (BMDM) infected with Salmonella enterica serovar Typhimurium, undergo highly significant reduction in refractive index, RI, compared to uninfected cells. Infected BMDM cells from genetically modified mice lacking an inflammatory protein that causes cell death, caspase 1, also exhibit similar decrease in RI. These data suggest that any reduction in RI of Salmonella ‐infected BMDMs is pathogen induced and independent of caspase 1‐induced inflammation or cell death. This finding suggests DHM may be useful for general real time monitoring of host cell interactions with infectious pathogens. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)