Connecting Proteins by Design. Cross-Linked Bis-Hemoglobin

Abstract

A new type of multifunctional reagent creates a specific connection within and between two hemoglobin tetramers, resulting in a cross-linked bis-tetramer (“CLBT”). The tetrafunctional reagent (N,N‘-5,5‘-bis[bis(3,5-dibromosalicyl)isophthalyl]terephthalamide, DBIT) was prepared by conversion of the corresponding tetraacid to the tetrakis(3,5-dibromosalicylate). Deoxy hemoglobin reacts with DBIT to give a cross-linked bis-tetramer as the major product. Patterns in tryptic digests reveal that there are modifications of amino groups at β-Lys-82 and β-Val-1, indicative of a structure in which each tetramer is cross-linked between these positions. The cross-linked bis-tetramer has a decreased affinity for oxygen and very low cooperativity in oxygen binding. The properties of the material provide insights into the nature of protein−protein interactions.

Functional Cross-Linked Hemoglobin Bis-tetramers: Geometry and Cooperativity

“…Reagent 1, with a central sulfone, gave the highest yield of a ∼128 kDa material (Table 1). Globin chain analysis (32) through C4 reverse-phase HPLC showed that only one component derived from native hemoglobin had been modified (Figure 2 (20) and counterparts isophthalyl phosphastes (23,24) that produced cross-linked hemoglobin bis-tetramers.…”

“…Since partial hydrolysis will lead to a more complex mixture of products, we chose to use dibromosalicylates to minimize partial destruction of the reactive portion of the reagents. The synthesis of acyl dibromosalicylate derivatives, which was achieved by reacting activated carboxylic acids with protected dibromosalicylic acid, gave useful yields of reagents (20). Although the acyl dibromosalicylates are less reactive than acyl phosphates and have additional steric limitations in their reactivity patterns, they efficiently cross-linked hemoglobins as bis-tetramers (20).…”

“…The synthesis of acyl dibromosalicylate derivatives, which was achieved by reacting activated carboxylic acids with protected dibromosalicylic acid, gave useful yields of reagents (20). Although the acyl dibromosalicylates are less reactive than acyl phosphates and have additional steric limitations in their reactivity patterns, they efficiently cross-linked hemoglobins as bis-tetramers (20). Therefore, we used tetrakis-dibromosalicylates (20) as the cross-linking reagents for this study.…”

“…Although the acyl dibromosalicylates are less reactive than acyl phosphates and have additional steric limitations in their reactivity patterns, they efficiently cross-linked hemoglobins as bis-tetramers (20). Therefore, we used tetrakis-dibromosalicylates (20) as the cross-linking reagents for this study. These new tetrakisdibromosalicylate cross-linking reagents were readily synthesized from commercial dicarboxylic acids, which were efficiently extended to relevant tetrakis-carboxylic acid precursors.…”

2001 Lemieux Award Lecture Organic chemistry and hemoglobin: Benefits from controlled alteration

“…Combining the reagent with hemoglobin gave material that had a molecular weight of about 128 kDa (by size exclusion chromatography based on standards from hemoglobin oligomers) (40). Polyacrylamide gel electrophoresis under nondenaturing conditions also gives the major peak at the same retention time as a 128 kDa standard.…”