Multivariate analysis of different insulin proteins
Stability and aggregation of insulin are studied using simultaneous fluorescence excitation-emission matrices (EEMs) and UV-Vis absorbance spectroscopy. Insulin is a protein-hormone produced by the pancreas and is necessary for basic metabolic processes. Commercial insulin therapeutics generally fall into two categories: short-acting and long-acting insulin. The difference between some short-acting and long-acting insulin lies, in some cases, in only one to three residues in the protein sequence. This variation, along with the controlled pH of storage and delivery, is used to either trigger or prevent the formation of insulin dimers and hexamers in the bloodstream. The formation of these aggregates allows the body to absorb insulin more slowly, while their absence leads to faster absorption.
Changes in protein stability and structure, such as those important to the pharmacokinetics of insulin, can potentially be measured using fluorescence emission spectra, UV-Vis absorbance spectra, or both, by leveraging intrinsically fluorescent amino acids. Traditionally, UV-Vis spectrophotometers and fluorometers are separate instruments. This application note presents a new and fast method for simultaneously generating individual excitation and emission spectra for all fluorescent sample components, providing information needed to correct the fluorescence spectra for sample concentration-dependent inner filter effects (IFE).
