Application of Western Blotting for the Eetection of Uncoupling Protein-2 (UCP-2) in Mitochondria from Smokers and Non-Smokers

Abstract

Introduction: Uncoupling proteins (UCPs) are a family of transmembrane anion transporters present in the inner mitochondrial membrane. UCP-2, which exhibits the widest distribution in various tissues, plays an important role in many physiological processes. Human UCP-2 studies have been hampered by the lack of a method for measuring this protein in an easily accessible human tissue, e.g., blood. The aim of this study was to develop such a method and test its utility by comparing UCP-2 levels in smokers and non-smokers. Material and Methods: Venous blood samples from 10 smoking and seven non-smoking volunteers were used for the study; lymphocytes were isolated employing Lymphoprep. UCP-2 levels were measured by Western blotting combined with chemoluminescence detection. Results: Total lymphocyte homogenates were found useless for measuring UCP-2 levels, but it was possible to measure UCP-2 in homogenates of purified lymphocyte mitochondria. There was a significant, though moderate, linear correlation between UCP-2 level and daily cigarette use. UCP-2 level in peripheral blood lymphocytes from smokers was higher than that in non-smokers. Conclusion: The method for measuring UCP-2 in peripheral blood lymphocytes opens the possibility of UCP-2 screening studies in humans and thus may be useful for studying the role of the protein in human physiology and pathology.