Physicochemical Characterization of Complex Drug Substances: Evaluation of Structural Similarities and Differences of Protamine Sulfate from Various Sources

Abstract

Abstract. The purpose of this study was to characterize and evaluate differences of protamine sulfate, a highly basic peptide drug, obtained from five different sources, using orthogonal thermal and spectroscopic analytical methods. Thermogravimetric analysis and modulated differential scanning calorimetry showed that all five protamine sulfate samples had different moisture contents and glass transition and melting temperatures when temperature was modulated from 25 to 270°C. Protamine sulfate from source III had the highest residual moisture content (4.7±0.2%) at 105°C, resulting in the lowest glass transition (109.7°C) and melting (184.2°C) temperatures compared with the other four sources. By Fourier-transform infrared (FTIR) spectroscopy, the five sources of protamine sulfate had indistinguishable spectra, and the spectra were consistent with a predominantly random coil conformation in solution and a minor population in a β-sheet conformation (~12%). Circular dichroism spectropolarimetry confirmed the FTIR results with prominent minima at 206 nm observed for all five sources. Finally, proton ( 1 H) nuclear magnetic resonance spectroscopy showed that all five protamine sulfate sources had identical spectra with backbone amide chemical shifts between 8.20 and 8.80 ppm, consistent with proteins with predominantly random coil conformation. In conclusion, thermal analyses showed differences in the thermal behavior of the five sources of protamine sulfate, while spectroscopic analyses showed the samples had a predominantly random coil conformation with a small amount of β-sheet present.

Facet-Specific Assembly of Proteins on SrTiO3 Polyhedral Nanocrystals

“…The band at 1242 cm −1 is consistent with the canonical values for β-sheet conformation2223. And the intense band at 1084 cm −1 can be attributed to the arginine of salmine24. By comparison with the spectra of salmine after adsorption on SrTiO 3 nanocrystals (Figure 3a), the shift of amide I adsorption band to 1655 cm −1 (α-helices), which is also attributed to the carbonyl stretching frequency to interaction with the surface25, and the diminishing of the bands at 1452 cm −1 and 1242 cm −1 as well as the increase in the ratios of amide I/amide II, suggest a strong interaction of salmine with SrTiO 3 nanocrystal and thus the conformational change of protein structure26.…”

Computational Approach to Elucidating Insulin–Protamine Binding Interactions and Dynamics in Insulin NPH Formulations

“…In the absence of the PDB structures of the four protamine peptides used in this study, their structure predictions were carried out using an AI-based program, AlphaFold. , Table shows the amino acid sequence used for the structure predictions . Random coiled structures for all four peptides were obtained, which is also in agreement with previous experimental results. , As insulin in the insulin NPH formulation exists as a hexamer, therefore, all our computational calculations were carried out on the insulin R6 hexamer structure (PDB ID: 1AIY ) downloaded from the RCSB Protein Data Bank. , These structures were further used for molecular docking and molecular dynamics simulations.…”

In vitro study of the specific interaction between poly(2-dimethylamino ethylmethacrylate) based polymers with platelets and red blood cells

“…With a secondary structure made from +/-of 12%  sheet (Awotwe-Otoo et al, 2012) protamine has obviously a more rigid and compact conformation compared to the random and flexible coil expected from the PDMAEMA (Cerda-Cristerna et al, 2011).…”