Bifunctional reagents and protein structure determination. The reaction of phenolic disulfonyl chlorides with lysozyme
✍ Scribed by David J. Herzig; Allan W. Rees; Richard A. Day
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 1964
- Tongue
- English
- Weight
- 657 KB
- Volume
- 2
- Category
- Article
- ISSN
- 0006-3525
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✦ Synopsis
It was established that phenol-2,Faisulfonyl chloride and a-naphthol-2,Faisulfonyl chloride introduce new intramolecular or tertiary bonds into lysozyme. The phenolic hydroxyl group seems to make the cross linking reagents soluble in aqueous media; it ww established that a sulfonylating species does dissolve prior to hydrolysis but that the chlorosulfonyl groups are hydrolyzed within seconds of dissolution. New covalently linked residues were shown t o be introduced by different spectra. Peptide maps of the lysozyme treated with pheno1-2,4-disulfonyl chloride] peroxidized, and digested with trypsin showed definite deletions and additions when compared with the control. Partial analysis of the peptides showed that cross linking between e-amino groups of lysine residues had occurred. A certain degree of stabilization of lysozyme to inactivation by certain inactivating conditions was noted after treatment with the two reagents. Ultracentrifugal analysis of the lysozyme treated with phenol-2,4disulfonyl chloride showed that there waa no detectable fraction having a higher molecular weight than native lysozyme. Tentative aasignments of positions of cross linking were made.
The reaction of bifunctional crosslinking reagents with proteins was first studied successfully by Zahn and his group. They investigated the reaction of bisdiazohexane, difluorodinitrobenzene12 and p,p'-difluorom,m'-dinitrodiphenyl sulfone3 with wool keratin, silk fibroin, and insulin, and found that these crosslinking reagents reacted with a-and e-amino, hydroxyl, and phenolic groups, among others. Wold4 studied the reaction of difluorodinitrodiphenyl sulfone with bovine serum albumin. He found that intramolecular crosslinking occurred. Broomfield and Scheraga5 studied the reaction of difluorodinitrobenzene with ribonuclease. Here they found that no crosslinking occurred, although there had been a reaction. This is probably due to the short distance between the reactive centers in the crosslinking reagent. Because the above reagents are insoluble in water, special methods must be used to eliminate the possibility