Use of Enzymes in Determining Structure

Enzymes can be used to give both qualitative and quantitative determinations, depending on the types of analyses made. Quantitative data can be obtained by the measurement of the increase in reducing values with time of hydrolysis and/or the determination of a specific product, such as glucose, using the glucose oxidase method of analysis (see section 3.5 in Chapter 3). Other specific products can be determined using qualitative and quantitative TLC or HPLC. 

Enzymes have played an important role in the determination of the structure of starch. Acid hydrolysis of starch provided some controversy about the possible presence of a-1 — 3, P-1 4, and P-1 6 linkages in starch. This was appar- 

Crystalline human salivary a-amylase produced 6 -phosphomaltotetraose from potato amylopectin, demonstrating that the covalently linked phosphate in potato starch was attached to the C-6 hydroxyl group by a phosphoester linkage in amylopectin [23]. 

Initially, the action of plant 3-amylase with amylose gave complete conversion to P-maltose [24], indicating that the amylose component of starch was a linear a-1 4-linked glucan. However, when highly purified, crystalline sweet potato 3-amylase was used, the amylose was only converted to 70% p-maltose [25-27]. 

The resistant fraction gave a deep blue color with triiodide, similar to that given by amylose. Treatment of the original amylose or the resistant fragment with pul-lulanase increased its conversion to 100% (3-maltose, indicating that some of the amylose molecules had a-1 6 branch linkages that prevented complete conversion to -maltose by (3-amylase [28,29]. 

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