Polyacrylic polyhydrazides as reagents for detection of glycoproteins

Glycoproteins immobilized on membranes can be detected with high selectivity and sensitivity by the four-step procedure described in this work. The glycoproteins are first oxidized by sodium periodate and then polyacrylic polyhydrazides are coupled to the aldehyde groups generated in the sugar part of the glycoproteins. In the third step, a glycoenzyme, such as horseradish peroxidase, is coupled to the remaining hydrazide groups on the polymer through the aldehydes formed in its glycan chains. In the last step, the visualization of glycoproteins is achieved through the reaction product of the bound glycoenzyme. The sensitivity of the glycoprotein detection is most critically dependent on the hydrazide reagent. Thus, dihydrazides were not satisfactory, a trihydrazide was better, and polyhydrazides were the best. Two different polyhydrazides were used. One was based on acrylamide and the other on N-acryloyl-tris(hydroxymethyl)aminomethane. The second one proved to be superior because it gave higher sensitivity with no detectable background staining. We have also investigated the influence of various reaction conditions on staining of glycoproteins having oligomannose and N-acetyllactosamine type glycan chains. Some of them, invertase and fetuin, could be detected with sensitivity similar to that of silver staining in gels and colloidal gold staining on the membranes. The detection of small quantities of Endo H-deglycosylated glycoproteins was possible under standard conditions only if several N-acetylglucosamine residues remained bound to the protein.