Changes of glucagon structure and function by ~(21)Ala site-directed mutagenesis

Glucagon gene mutation was testified by DNA sequencing. After expression, ~(21)Ala-glucagon was purified by affinity chromatography and C8 RP-HPLC. ~(21)Ala-glucagon was identified by relative molecular mass assay with MS. The comparison between the secondary structures of recombinant glucagon and ~(21)Ala-glucagon by CD indicated that alpha-helix content increased in ~(21)-Ala-glucagon. By means of rabbit blood glucose test after glucagon or ~(21)Ala-glucagon treatment, it showed that the biological activity of ~(21)Ala-glucagon was 51% less than that of glucagon (P<0.01). The results suggest that ~(21)Asp has no relationship with alpha-helix formation of glucagon secondary structure, but it may be very important in glucagon biological activity. Because ~(21)Asp may act as a binding site of Ca~(2+), it will promote the binding of glucagon and glucagon receptor with Ca~(2+) participation.