Biochemistry - Chemical Reactions in Living Cells, Volume 1 - D. Metzler 1980

The molecules we are made of
How we study molecular structure
Methods based on end-group labeling

In 1957, F. Sanger was awarded the Nobel Prize in Chemistry for determining the Introduction/19.html">Primary Structure of Insulin. It was the first protein whose Amino Acid Sequence was successfully established; Sanger dedicated 10 years to this work. His approach was based on the partial Hydrolysis of peptide chains that had been previously labeled:

Class="center">

via the reaction of a free amino group with fluorodinitrobenzene [equation (2-27)].

The bond with the dinitrophenyl group is acid-stable, and therefore complete acid hydrolysis of the labeled peptide released the dinitrophenylated amino acid (a yellow-colored compound) that was previously located at the N-terminus of the chain. In addition, Sanger utilized labeled ε-amino groups of Lysine residues. Partial Acid Hydrolysis of the labeled Peptides in this case led to The formation of small fragments, for which the Amino Acid Composition was subsequently determined. Finally, Sanger pieced together the resulting amino acid puzzle and established The sequence of the two chains of the insulin molecule, containing 21 and 30 residues respectively, which are linked together in the intact molecule by disulfide bridges (Fig. 4-13). In recent years, dansyl chloride has been more commonly used instead of fluorodinitrobenzene. The resulting peptide derivatives are strongly fluorescent, making it possible to perform end-group analysis with significantly smaller amounts of peptides.

There are several Methods for labeling the SH groups of protein side chains. One of the most widespread is based on The Use of Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid), DTNB [equation (2-28)].

This reagent reacts quantitatively with —SH groups, forming mixed disulfides and releasing a thiol anion that absorbs light at 412 nm, which allows the —SH group content in the protein to be determined. Unfortunately, this can be accompanied by parallel disulfide exchange reactions involving additional molecules of DTNB, leading to the Cleavage of disulfide Bonds in the protein [143]. Besides those mentioned above, there are numerous Other Reagents that allow the modification of various side groups within the protein molecule [138, 143a].

In the case of CARBOHYDRATES, the classical method for identifying end groups is exhaustive methylation. Repeated Treatment with a methylating reagent, such as dimethyl sulfate, converts all free OH groups into OCH₃ groups. Complete acid hydrolysis followed by the Separation of the methylated sugars and their quantitative determination makes it possible to estimate the number of terminal units (containing four methoxy groups), the number of units in unbranched chain segments (containing three methoxy groups each), and the number of branch points (containing two methoxy groups each). Furthermore, The structure of the methylated derivatives provides information on the arrangement of linkages within the sugar rings.

A relatively new method for labeling the reducing ends of carbohydrate chains is the reduction of aldehyde groups with sodium borotritide (NaB³H₄). The radioactive label introduced in this way makes it possible, using chromatogram radioautographs, to determine THE POSITION OF fragments cleaved from the reducing ends.

One of the most valuable reagents used to determine carbohydrate structure is periodic acid (or its sodium salt, sodium periodate). Under the action of periodic acid, C—C bonds flanked on both sides by OH groups are cleaved, resulting in the formation of dialdehydes.

The method allows for quantitative measurements. A few hours after THE START OF the reaction, the excess periodate is removed with Ethylene glycol; The amount of periodate consumed during the oxidation process can also be determined. If the hydroxyl groups are attached to three consecutive carbon atoms, the central atom is cleaved off as formic acid, the amount of which can likewise be accurately determined. In addition, after removing the excess periodate, the dialdehyde can be reduced by adding solid sodium borohydride to form stable CH₂OH groups, and subsequently cleaved via mild acid hydrolysis of the acyclic acetal bonds, followed by the separation and Modification of the resulting fragments. This sequence of reactions is referred to as Smith degradation [144].

An example of the combined use of several Methods for determining the structure of a complex polysaccharide is given in Table 2-12.

The Terminal Groups of polyribonucleotides are also cleaved with periodate after first removing the phosphate groups at the 3'-hydroxyls using phosphomonoesterase. The resulting dialdehydes are then reduced with sodium borotritide. Another method for incorporating a radioactive terminal group into polynucleotides is based on The transfer of a phosphoryl group from ATP molecules under the action of nonspecific polynucleotide kinase, yielding a free 5'-hydroxyl group on the polyribo- or polydeoxyribonucleotide [145].

A valuable method for studying DNA Structure is the nearest-neighbor frequency analysis. Developed by A. Kornberg and co-workers, it consists of the following [146]. As precursors for DNA Synthesis, a single radioactive ³²P-containing nucleoside triphosphate is taken [in equation (2-30), this is deoxyadenosine triphosphate], while the other three

Table 2-12 Structure Determination of the 6-O-methylglucose-containing lipopolysaccharide from Mycobacterium phlei^a,b

1. This bond is selectively cleaved by α-amylase.

2. Partial hydrolysis yields a series of 6-O-methylglucose oligomers.

3. Cleavage of D-glyceric acid and reduction of the resulting end group with NaB³H₄ allows a set of radioactive fragments to be obtained during subsequent chain cleavage (via partial acid hydrolysis).

4. Conversion into an ester and reduction with NaB³H₄ to an alcohol (glycerol) makes it possible, after partial acid hydrolysis, to obtain another set of radioactive fragments.

5. Methylation establishes the presence of branching, whereas propylation of non-reducing terminal groups determines the position of glucose and 3-methylglucose.

6. According to another Procedure, O-acyl groups were replaced by —OCH₃. Subsequent periodate oxidation, reduction with NaB³H₄, and hydrolysis (Smith degradation) led to the formation of methylated fragments, the identification of which made it possible to determine the positions of the original acyl groups.

^а Saier М. Н., Jr., Ballou С. Е., J. Biol. Chem., 243, 4332, 1968; Smith W. L., Ballou C. E., J. Biol. Chem., 248, 7118—7125, 1973; Gray G. R., Ballou C. E., J. Biol: Chem., 247, 8129—8135, 1972.

б The positions marked with a filled triangle contain Fatty acids: 3 acetyls, 1 propionyl, 1 isobutyryl, and 1 octanoyl; the positions marked with an open triangle contain succinyl groups. Methyl groups are indicated by a filled circle.

nucleoside triphosphate is left unlabeled. In the presence of DNA polymerase, which catalyzes chain elongation, the primer DNA strand extends from the 3'-end (Chap. 15, Sec. A.3.a). The template strand present [not shown in equation (2-30)] ensures the incorporation into the growing chain of the exact nucleotide required to form the Watson-Crick double-helical structure. The chemical mechanism of this process will be discussed later (Chap. 15, Sec. A.3.a) and is irrelevant here. The key point is that ³²P ends up in the phosphodiester bridge that links the nucleotide originally containing ³²P to the 3'-hydroxyl group of the adjacent nucleotide.

The ³²P-containing product is subsequently hydrolyzed using micrococcal nuclease and Spleen phosphodiesterase (Table 2-11) to yield the fragments shown in equation 2:30. Since these hydrolytic Enzymes catalyze _L only b-type cleavages (Table 2-11), ³²P is now incorporated into the nucleotide that was the nearest neighbor of adenosine in the DNA structure. By measuring the radioactivity of the 3'-NUCLEOTIDES of adenine, thymine, cytosine, and guanine, one can determine the frequencies of AA, TA, CA, and GA pairs in DNA. Using other radioactive nucleoside triphosphates (one type per experiment), such frequencies are obtained for all possible pairs. The results of the experiment described above demonstrate that the strands of The Double Helix are indeed antiparallel, as predicted by Watson and Crick (for details, see Davidson [146]); otherwise, the nearest-neighbor frequencies would have been different.

Similarly, but using alkali-catalyzed b-type cleavage, the frequencies of various pairs were determined in the RNA chain synthesized on a DNA template [145].