From the foregoing discussion, it should be evident that while the assay of limit dextrinase can be difficult, the use of dyed pullulan has been a significant advance. However, just as important as assaying the enzyme correctly is the correct extraction of the enzyme. It is thought that in vivo in germinating barley, the enzyme exists in three forms—bound and inactive, soluble and active (known as "free"), and soluble and inactive (42,

43). Furthermore, limit dextrinase inhibitors have been detected in barley grain extracts (44). While barley grains at maturity have a high level of limit dextrinase inhibitor, the level is lower in wheat, and the inhibitor is absent in rice and maize (45). All these factors complicate the interpretation of limit dextrinase activity measurements. However, it is currently believed that the "total" activity of limit dextrinase in germinating barley grain can be determined by extraction for 5 h with a reducing agent, whereas extraction for 5 h without a reducing agent provides a measure of the "free" activity (soluble and active).

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