Protein stability remains one of the main factors limiting the realization of the full potential of protein therapeutics. Poly(ethylene glycol) (PEG) conjugation to proteins has evolved into an important tool to overcome instability issues associated with proteins. The observed increase in thermodynamic stability of several proteins upon PEGylation has been hypothesized to arise from reduced protein structural dynamics, although experimental evidence for this hypothesis is currently missing. To test this hypothesis, the model protein α-chymotrypsin (α-CT) was covalently modified with PEGs with molecular weights (MW) of 700, 2000 and 5000 and the degree of modification was systematically varied. The procedure did not cause significant tertiary structure changes. Thermodynamic unfolding experiments revealed that PEGylation increased the thermal transition temperature (Tm) of α-CT by up to 6°C and the free energy of unfolding (ΔGU (25°C)) by up to 5 kcal/mol. The increase in stability was found to be independent of the PEG MW and it leveled off after an average of four PEG molecules were bound to α-CT. Fourier-transformed infrared (FTIR) H/D exchange experiments were conducted to characterize the conformational dynamics of the PEG-conjugates. It was found that the magnitude of thermodynamic stabilization correlates with a reduction in protein structural dynamics and was independent of the PEG MW. Thus, the initial hypothesis proved positive. Similar to the thermodynamic stabilization of proteins by covalent modification with glycans, poly(ethylene glycol) thermodynamically stabilizes α-CT by reducing protein structural dynamics. These results provide guidance for the future development of stable protein formulations.

Purpose This study determined (1) how many vessels (i.e. the vessel sampling) are needed to reliably characterize the bulbar conjunctival microvasculature and (2) if characteristic information can be obtained from the distribution histogram of the blood flow velocity and vessel diameter. Methods Functional Slit-lamp Biomicroscope was used to image hundreds of venules per subject. The bulbar conjunctiva in 5 healthy human subjects was imaged on 6 different locations in the temporal bulbar conjunctiva. The histograms of the diameter and velocity were plotted to examine whether the distribution was normal. Standard errors were calculated from the standard deviation and vessel sample size. The ratio of the standard error of the mean over the population mean was used to determine the sample size cutoff. The velocity was plotted as a function of the vessel diameter to display the distribution of the diameter and velocity. Results The results showed that the sampling size was approximately 15 vessels, which generated a standard error equivalent to 15% of the population mean from the total vessel population. The distributions of the diameter and velocity were unimodal, but also somewhat positively skewed and not normal. The blood flow velocity was related to the vessel diameter (r =0.23, P < 0.05). Conclusion This was the first study to determine the sampling size of the vessels and the distribution histogram of the blood flow velocity and vessel diameter, which may lead to a better understanding of the human microvascular system of the bulbar conjunctiva.