Reversible reductive alkylation of amino groups in proteins

Biochemistry ◽  
1979 ◽  
Vol 18 (24) ◽  
pp. 5392-5399 ◽  
Author(s):  
Kieran F. Geoghegan ◽  
Donna M. Ybarra ◽  
Robert E. Feeney
Keyword(s):  
Reductive Alkylation ◽  
Amino Groups

Selective fluorescent labeling of amino groups of bovine pancreatic tryspin inhibitor by reductive alkylation

Biopolymers ◽  
1986 ◽  
Vol 25 (9) ◽  
pp. 1645-1658 ◽  
Author(s):  
Dan Amir ◽  
Daniel P. Levy ◽  
Yehuda Levin ◽  
Elisha Haas
Keyword(s):  
Fluorescent Labeling ◽  
Reductive Alkylation ◽  
Amino Groups

Conjugation of N-acylated amino sugars to protein by reductive alkylation using sodium cyanoborohydride: application to an azo derivative of α-amanitin

1991 ◽  
Vol 69 (7) ◽  
pp. 418-427 ◽  
Author(s):  
Jerald E. Mullersman ◽  
James F. Preston III
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Protein Modification ◽  
Reductive Coupling ◽  
Reductive Alkylation ◽  
Amino Groups ◽  
Protein Polymerization ◽  
Calf Thymus ◽  
Aldehyde Groups

Reductive alkylation mediated by cyanoborohydride is an attractive approach to the conjugation of small molecules, such as drugs, to proteins. This reaction is specific for protein amino groups and can be conducted under mild conditions with little risk of protein polymerization. However, the lability of the aldehyde function that is needed in such reactions presents a difficulty. We have investigated the use of derivatives of D-galactosamine and D-glucosamine in reductive alkylation, since these sugars contain aldehyde groups that are inherently protected and that may be readily linked to other molecules through their amino groups. The amino groups of these sugars were acylated with N-4-nitrobenzoylglycylglycine. Studies of the reductive coupling of the resultant adducts to bovine serum albumin revealed that conjugation to albumin is strongly dependent on cyanoborohydride, is much faster in the presence of borate, and shows a marked increase in rate between pH 7.0 and 9.0. In the presence of borate, the glucosamine derivative coupled much more rapidly than did the galactosamine derivative. The aryl nitro group of the glucosamine adduct was selectively reduced to an amine, diazotized, and reacted with α-amanitin to form an azo compound. This azo derivative was reductively coupled to form conjugates that inhibit calf thymus RNA polymerase II.Key words: α-amanitin, borate, bovine serum albumin, cyanoborohydride, protein modification, reductive alkylation.

Reductive alkylation of amino groups in proteins

Biochemistry ◽  
1968 ◽  
Vol 7 (6) ◽  
pp. 2192-2201 ◽  
Author(s):  
Gary E. Means ◽  
Robert E. Feeney
Keyword(s):  
Reductive Alkylation ◽  
Amino Groups

Aldehyde gold clusters for molecular labeling

1995 ◽  
Vol 53 ◽  
pp. 858-859
Author(s):  
James F. Hainfeld ◽  
Frederic R. Furuya
Keyword(s):  
Covalent Bond ◽  
Aldehyde Group ◽  
Gold Clusters ◽  
Side Reactions ◽  
Amino Groups ◽  
Sodium Cyanoborohydride ◽  
Schiff’S Base ◽  
Protein Molecules ◽  
Hydroxysuccinimide Esters ◽  
Primary Amino

Glutaraldehyde is a useful tissue and molecular fixing reagents. The aldehyde moiety reacts mainly with primary amino groups to form a Schiff's base, which is reversible but reasonably stable at pH 7; a stable covalent bond may be formed by reduction with, e.g., sodium cyanoborohydride (Fig. 1). The bifunctional glutaraldehyde, (CHO-(CH2)3-CHO), successfully stabilizes protein molecules due to generally plentiful amines on their surface; bovine serum albumin has 60; 59 lysines + 1 α-amino. With some enzymes, catalytic activity after fixing is preserved; with respect to antigens, glutaraldehyde treatment can compromise their recognition by antibodies in some cases. Complicating the chemistry somewhat are the reported side reactions, where glutaraldehyde reacts with other amino acid side chains, cysteine, histidine, and tyrosine. It has also been reported that glutaraldehyde can polymerize in aqueous solution. Newer crosslinkers have been found that are more specific for the amino group, such as the N-hydroxysuccinimide esters, and are commonly preferred for forming conjugates. However, most of these linkers hydrolyze in solution, so that the activity is lost over several hours, whereas the aldehyde group is stable in solution, and may have an advantage of overall efficiency.

The Action of Thrombin on Modified Fibrinogen

1983 ◽  
Vol 49 (03) ◽  
pp. 208-213
Author(s):  
A J Osbahr
Keyword(s):  
Physical Properties ◽  
Negative Charge ◽  
Lysine Residue ◽  
Fibrinopeptide A ◽  
Amino Groups ◽  
Lysine Residues ◽  
Citraconic Anhydride

SummaryThe modification of canine fibrinogen with citraconic anhydride modified the ε-amino groups of the fibrinogen and at the same time generated additional negative charges into the protein. The addition of thrombin to the modified fibrinogen did not induce polymerization; however, the fibrinopeptide was released at a faster rate than from the unmodified fibrinogen. The physical properties of the citraconylated fibrinogen were markedly altered by the modification of 50-60 lysine residues in one hour. A modified fibrinopeptide-A was released by thrombin from the modified fibrinogen and was electrophoretically more anionic than the unmodified fibrinopeptide-A. Edman analysis confirmed the modification of the lysine residue present in the peptide. The rate of removal of citraconylated fibrinopeptide-A from modified fibrinogen by thrombin was 30 to 40 percent greater than the cleavage of unmodified fibrinopeptide-A from unmodified fibrinogen. However, the modification of 60 or more lysine residues in the fibrinogen produced a decrease in the rate of cleavage of citraconylated fibrinopeptide-A. The results suggest that additional negative charge in the vicinity of the attachment of fibrinopeptide-A to canine fibrinogen aids in the removal of the peptide by thrombin.

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