Detection of Proteins in Starch Granule Channels

Proteins were detected in channels of commercial starches of normal maize, waxy maize, sorghum, and wheat through labeling with a protein‐specific dye and examination using confocal laser scanning microscopy (CLSM). The dye, specifically 3‐(4‐carboxybenzoyl)quinoline‐2‐carboxaldehyde (CBQCA), fluoresces only after it reacts with primary amines in proteins, and CLSM detects fluorescence‐labeled protein distribution in an optical section of a starch granule while it is still in an intact state. Starch granules in thin sections of maize kernels also had channel proteins, indicating that proteins are native to the channels and not artifacts of isolation. Incubation of maize starch with protease (thermolysin) removed channel proteins, showing that channels are open to the external environment. SDS‐PAGE analysis of total protein from gelatinized commercial waxy maize starch revealed two major proteins of about M_r 38,000 and 40,000, both of which disappeared after thermolysin digestion of raw starch. Commercial waxy maize starch granule surface and channel proteins were extracted by SDS‐PAGE sample buffer without gelatinization of the granules. The major M_r 40,000 band was identified by MALDI‐TOF‐MS and N‐terminal sequence analysis as brittle‐1 (bt1) protein.