Modification of tryptophan residues in immunoglobulin M by 2-hydroxy-5-nitrobenzyl bromide

The modification of tryptophan residues in monoclonal immunoglobulin M (IgM) by 2-hydroxy-5-nitrobenzyl bromide (RK) was studied at pH 2.0-2.85 and 7.0 and a RK to tryptophan molar ratio (K) from 1 to 40. At pH 2.85, the number of RK residues bound to IgM (N) in account to one HL-fragments does not exceed 10 (the HL-fragment of IgM contains 14 tryptophan residues); the plot of N vs K reaches a plateau at K greater than 20. When the pH is lowered to approximately 2, N rises to approximately 15, but the plateau is not reached. At pH 7.0, the modified IgM with N greater than 1 gives a sediment, while the product with N approximately equal to 1 remains in solution. Evidently, the latter contains the most accessible tryptophan residue (calculated per one HL-fragment). This residue was found to be one of the three residues localized in the C mu 2-domain and the adjoining N-terminal part. The possibility of multiple modification of tryptophan residues during the RK interaction with IgM in acid medium at high values of K is discussed.     

Chemical modification of tryptophan residues of leucyl tRNA synthetase by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide

The structural accessibility of tryptophan residues in leucyl-tRNA synthetase from cow mammary gland has been studied using chemical modifications by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide. The modifications were monitored by UV absorbance and intrinsic fluorescence of the enzyme's tryptophan residues. Under native conditions, at pH 7,8, only two exposed tryptophan residues are modified in each subunit of the dimeric enzyme. Under denaturing conditions, in 6 M guanidine hydrochloride solution, internal tryptophan residues are also modified as a consequence of unfolding of the native tertiary structure of the enzyme. Modifications of tryptophan residues resulted in inactivation of leucyl-tRNA synthetase both in aminoacylation and ATP-PPi exchange reactions. In the specific complex of leucyl-tRNA synthetase with the cognate tRNALeu one of exposed tryptophan residues is protected by tRNALeu and is not modified by the above reagents.     

The chemistry of the reaction of 2-hydroxy-5-nitrobenzyl bromide with his-32 of alpha-lactalbumin.

His-32 of bovine or human alpha-lactalbumin reacts with the tryptophan reagent 2-hydroxy-5-nitrobenzyl bromide at pH 7. The reaction depends on the native conformation of the alpha-lactalbumin molecule and it is restricted to position 1 of the imidazole nucleus. The synthesis and characterization of 1-(2-hydroxy-5-nitrobenzyl)-histidine, 3-(2-hydroxy-5-nitrobenzyl)-histidine and 1,3-bis(2-hydroxy-5-nitrobenzyl)-histidine are described.     

Spectrophotometric estimation of protein concentration in the presence of tryptophan modified by 2-hydroxy-5-nitrobenzyl bromide

A spectrophotometric method makes it possible to determine the concentration of a protein after covalent modification of tryptophan residues by 2-hydroxy-5-nitrobenzyl bromide. Molar absorption coefficients for the 2-hydroxy-5-nitrobenzyl chromophore, reported here in the pH range from 4.0 to 10.9, can be used to correct the protein absorbance values at 280 nm, which then provides the basis for calculating protein concentration in the usual way. The method was tested with alpha-lactalbumin, beta-lactoglobulin, pepsin, and soybean trypsin inhibitor; spectrophotometrically estimated concentrations of these proteins agreed closely with values obtained by amino acid analysis.     

Kinetic studies on mammalian cytochrome c modified with 2-hydroxy-5-hydroxy-5-nitrobenzyl bromide.

The reduction of 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c by the chromous ion was studied by stopped-flow techniques. At pH6.5 the reduction of 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c is complex, showing the presence of three distinct phases. Two chromium concentration-dependent phases are observed (1.1 X 10(5) M-1-S-1, phase 1; 1.25 X 10(4)M-1-S-1, phase 2) and one slow first-order process (0.25S-1, phase 3). A comparison of the static and kinetic difference spectra, along with the data from the reduction of the reoxidized reduced protein, suggests that the slow chromium concentration-independent phase is due to a slow conformational event after fast reduction of the NO2 group. The rates of the chromium concentration-dependent phases show a marked variation with pH above 7.5. The activation energies for the three processes were also measured at 33.2, 38.6 and 69.7 kJ-mol-1 for phases 1, 2 and 3 respectively. The reaction of reduced 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c with CO was foollowed by means of both stopped-flow and flash photolysis. The combination with CO at pH 6.8 as measured in stopped-flow experiments showed two phases, one CO-dependent phase (phase 2, 2.4 X 10(2)M-1-S-1) and one CO-independent phase (phase 1, 0.015S-1). Investigation of the pH-dependence of the phases showed both the rates and amounts of each phase to be pH-invariant. CO recombination, after photolytic removal, was found to be biphasic; a CO-dependent phase (phase 2, 2.4 X 10(2)M-1-S-1) and a CO-independent phase (phase 1, 1.0s-1) were observed. A tentative model which can accommodate these observations is proposed.     

Studies on the effect of 2-hydroxy-5-nitrobenzyl bromide on the mechanism of insulin.

The effects of HNB were tested on the binding of insulin to fat cells and fat cell membranes as well as the responsiveness of fat cells to insulin. Although metabolism was found to be inhibited, the reagent was found to stimulate the binding of insulin. The stimulatory effect on insulin binding as well as inhibitory effect on metabolism was reversed by free tryptophan consumption of the reagent. The results are discussed relative to these findings.     

The role of tryptophanyl residues in heavy meromyosin as studied by chemical modification with 2-hydroxy-5-nitrobenzyl bromide.

1. Two moles of 2-hydroxy-5-nitrobenzyl group bound selectively to one mole of heavy meromyosin when it was treated with 2-hydroxy-5-nitrobenzyl bromide, a specific reagent for tryptophanyl residues. The binding with ADP, the size of the initial burst of Pi liberation and the difference absorption spectrum with and without ADP of the bound 2-hydroxy-5-nitrobenzyl groups were measured with heavy meromyosin modified with various amounts of reagent. The properties of the modified heavy meromyosin did not change until the molar binding ratio of the reagent, rH, was about 1, but the properties changed remarkably when rH increased from 1 to 2. 2. Subfragment-1 was prepared from the modified heavy meromyosin by trypsin [EC 3.4.21.4] digestion. The molar binding ratio of the reagent in subfragment-1, rS, was found to be less than 0.1 when rH of the starting heavy meromyosin was less than 0.8. However, rS was about 0.5 in subfragment-1 prepared from heavy meromyosin of rH about 2. The results indicate that only one mole of 2-hydroxy-5-nitrobenzyl group, which was bound with lower reactivity than the other, was bound to a head part of heavy meromyosin. 3. Subfragment-1 fraction prepared from the modified heavy meromyosin could be separated into two fractions by DE-32 cellulose column chromatography; the subfragment-1 portion which eluted later showed a higher rS than that eluted in front. The binding with ADP, the size of the initial burst of Pi liberation and the difference absorption spectrum induced by ATP were measured with the modified subfragment-1 separated by DE-32 cellulose column chromatography. The ADP-binding ability and the size of the initial burst were not dependent on rS, and coincided with those of subfragment-1 prepared from unmodified heavy meromyosin. 4. The results of ADP binding studies suggest that heavy meromyosin is constituted from nonidentical subunits, and that there is an interaction between them which controls the ADP binding. Two tryptophanyl residues having specific reactivity toward 2-hydroxy-5-nitrobenzyl bromide are assumed to be involved in the interaction.