Isolation and Characterization of Soluble Proteins in Grapes, Grape Juice, and Wine

Abstract

Improved sensitive techniques for the determination of soluble proteins in Gewürztraminer and White Riesling grapes, juice, and wine are reported. These techniques (1) minimize interference from phenolics and oxidase enzymes during extraction of proteins, (2) permit direct determination of proteins in juice and wine samples by electrophoretic techniques without prior pretreatment and concentration, (3) permit the detection of glycoprotein, and (4) permit the simultaneous determination of protein molecular weight (MW) and isoelectric point (pl) by two-dimensional isoelectric focusing-lithium dodecyl sulfate polyacrylamide gel electrophoresis (IF-LDS PAGE). Phenolic scavengers, Amberlite XAD-4 and polyvinylpolypyrrolidone (PVPP) and the enzyme inhibitors, phenylmethylsulfonyl fluoride (PMSF) and diethyldithiocarbamate (DECA), are used for protein isolation. One-dimensional 7.5% to 15% sucrose-stabilized linear gradient LDS PAGE with silver staining permits effective resolution of 3 µg of protein. The sensitivity for protein detection is increased 100-fold (detection at 10 ng level) by using silver staining instead of Coomassie blue. Changes in relative concentration of proteins on stained gels were determined by laser scanning densitometry. Two-dimensional IF-LDS PAGE, as well as protein blotting for glycoprotein detection, were used for further separation and characterization of the soluble proteins in wine. Grapes contain up to 41 protein fractions with MW in the range of 11 200 to 190 000; juices and wines from these grapes contain up to 25 protein fractions with MW in the range of 11 200 to 65 000. Each of these fractions were further separated into several fractions on the basis of their pl. A high proportion of wine protein fractions is of low pl (4.1 - 5.8) and low MW (20 000 - 30 000). Three protein fractions with MW of 12 600, 25 000, and 28 000 are identified as containing glycoproteins in Gewürztraminer wine. Only two fractions, with MW of 25 000 and 28 000, were detected as containing glycoproteins in White Riesling wine.